Therapeutic Monoclonal Antibodies that Neutralize Botulinum Neurotoxins

ABSTRACT

This invention provides antibodies that specifically bind to and typically neutralize  botulinum  neurotoxins (e.g., BoNT/A, BoNT/B, BoNT/E, etc.) and the epitopes bound by those antibodies. The antibodies and derivatives thereof and/or other antibodies that specifically bind to the neutralizing epitopes provided herein can be used to neutralize  botulinum  neurotoxin and are therefore also useful in the treatment of botulism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of and priority to U.S. Ser. No.60/896,332, filed on Mar. 22, 2007, and U.S. Ser. No. 60/942,173, filedon Jun. 5, 2007, both of which are incorporated herein by reference intheir entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support by Grant No: U01AI056493, awarded by the National Institutes of Health, and byDepartment of Defense Grant DAMD17-98-C-8030. The Government of theUnited States of America has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates antibodies that neutralize botulinum neurotoxins(e.g., BoNT/A) and their use in the treatment of botulism.

BACKGROUND OF THE INVENTION

Botulism is caused by botulinum neurotoxin secreted by members of thegenus Clostridium and is characterized by flaccid paralysis, which ifnot immediately fatal requires prolonged hospitalization in an intensivecare unit and mechanical ventilation. Naturally occurring botulism isfound in infants or adults whose gastrointestinal tracts becomecolonized by Clostridial bacteria (infant or intestinal botulism), afteringestion of contaminated food products (food botulism), or in anaerobicwound infections (wound botulism) (Center for Disease Control (1998)Botulism in the United States, 1899-1998. Handbook for epidemiologists,clinicians, and laboratory workers. Atlanta, Ga. U.S. Department ofHealth and Human Services, Public Health Service: downloadable at“www.bt.cdc.gov/agent/botulism/index.asp”). Botulism neurotoxins (BoNTs)are also classified by the Centers for Disease Control (CDC) as one ofthe six highest-risk threat agents for bioterrorism (the “Category Aagents”), due to their extreme potency and lethality, ease of productionand transport, and need for prolonged intensive care (Amon et al. (2001)JAMA 285: 1059-1070). Both Iraq and the former Soviet Union producedBoNT for use as weapons (United Nations Security Council (1995) Tenthreport of the executive committee of the special commission establishedby the secretary-general pursuant to paragraph 9(b)(I) of securitycouncil resolution 687 (1991), and paragraph 3 of resolution 699 (1991)on the activities of the Special Commision; Bozheyeva et al. (1999)Former soviet biological weapons facilities in Kazakhstan: past,present, and future. Center for Nonproliferation Studies, MontereyInstitute of International Studies), and the Japanese cult Aum Shinrikyoattempted to use BoNT for bioterrorism (Arnon et al. (2001) supra). As aresult of these threats, specific pharmaceutical agents are needed forprevention and treatment of intoxication.

No specific small molecule drugs exist for prevention or treatment ofbotulism, but an investigational pentavalent toxoid vaccine is availablefrom the CDC (Siegel (1988) J. Clin. Microbiol. 26: 2351-2356) and arecombinant vaccine is under development (Smith (1998) Toxicon 36:1539-1548). Regardless, mass civilian or military vaccination isunlikely due to the rarity of disease or exposure and the fact thatvaccination would prevent subsequent medicinal use of BoNT.Post-exposure vaccination is useless, due to the rapid onset of disease.Toxin neutralizing antibody (Ab) can be used for pre- or post-exposureprophylaxis or for treatment (Franz et al. (1993) Pp. 473-476 In B. R.DasGupta (ed.), Botulinum and Tetanus Neurotoxins: Neurotransmission andBiomedical Aspects. Plenum Press, New York). Small quantities of bothequine antitoxin and human botulinum immune globulin exist and arecurrently used to treat adult (Black and Gunn. (1980) Am. J. Med., 69:567-570; Hibbs et al. (1996) Clin. Infect. Dis., 23: 337-340) and infantbotulism (Arnon (1993). Clinical trial of human botulism immuneglobulin, p. 477-482. In B. R. DasGupta (ed.), Botulinum and TetanusNeurotoxins: Neurotransmission and Biomedical Aspects. Plenum Press, NewYork) respectively.

Recombinant monoclonal antibody (mAb) could provide an unlimited supplyof antitoxin free of infectious disease risk and not requiring humandonors for plasmapheresis. Given the extreme lethality of the BoNTs,mAbs must be of high potency in order to provide an adequate number ofdoses at reasonable cost. The development of such mAbs has become a highpriority research aim of the National Institute of Allergy andInfectious Diseases. While to date no single highly potent mAbs havebeen described, we recently reported that combining two to three mAbscould yield highly potent BoNT neutralization (Nowakowski et al. (2002)Proc. Natl. Acad. Sci. USA, 99: 11346-50).

The development of mAb therapy for botulism is complicated by the factthat there are at least seven BoNT serotypes (A-G) (Hatheway (1995)Curr. Top. Microbio. Immunol, 195: 55-75.) that show little, if any,antibody cross-reactivity. While only four of the BoNT serotypesroutinely cause human disease (A, B, E, and F), there has been onereported case of infant botulism caused by BoNT C (Oguma et al. (1990)Lancet 336: 1449-1450), one outbreak of foodborne botulism linked toBoNT D (Demarchi, et al. (1958) Bull. Acad. Nat. Med., 142: 580-582),and several cases of suspicious deaths where BoNT G was isolated(Sonnabend et al. (1981) J. Infect. Dis., 143: 22-27). AerosolizedBoNT/C, D, and G have also been shown to produce botulism in primates bythe inhalation route (Middlebrook and Franz (1997) Botulinum Toxins,chapter 33. In F. R. Sidell, E. T. Takafuji, D. R. Franz (eds.), MedicalAspects of Chemical and Biological Warfare. TMM publications,Washington, D.C.), and would most likely also affect humans. Thus it islikely that any one of the seven BoNT serotypes can be used as abiothreat agent.

Variability of the BoNT gene and protein sequence within serotypes hasalso been reported and there is evidence that such variability canaffect the binding of monoclonal antibodies to BoNT/A (Kozaki et al.(1998) Infect. Immun., 66: 4811-4816; Kozaki et al. (1995) Microbiol.Immunol., 39: 767-774).

SUMMARY OF THE INVENTION

This invention pertains to antibodies that bind to and neutralizebotulinum neurotoxin(s). We have discovered that particularly effectiveneutralization of a Botulism neurotoxin (BoNT) serotype can be achievedby the use of neutralizing antibodies that bind two or more subtypes ofthe particular neurotoxin serotype with high affinity and/or bycombinations of such antibodies. In certain embodiments this inventionprovides improved antibodies that bind BoNT subtypes BoNT/A, BoNT/B, andBoNT/E. In certain embodiments this invention provides for compositionscomprising neutralizing antibodies that bind two or more BoNT subtypes(e.g., BoNT/A1, BoNT/A2, BoNT/A3, etc.) with high affinity.

In certain embodiments this invention provides a neutralizing antibodyfor Botulinuym neurotoxin (BoNT). The antibody typically comprises atleast one VH complementarity determining region (CDR) selected from thegroup consisting of a 2A10 VH CDR, a 3E1VH CDR, a 3E2VH CDR, a 3E3VHCDR, a 3E4VH CDR, a 3E4.1VH CDR, a 3E5VH CDR, a 3E6VH CDR, a 3E6.1VHCDR, a 4E11VH CDR, a 4E13VH CDR, a 4E16VH CDR, a 4E16.1VH CDR, a 4E17VHCDR, a 4E17.1VH CDR, an A12 VH CDR, a 6A12 VH CDR, a B1.1 VH CDR, a B6VH CDR, a B6.1 VH CDR, a B8 VH CDR, a B8.1 VH CDR, a B11 VH CDR, a B11C3VH CDR, a B11E8 VH CDR, a B12 VH CDR, a B12.1 VH CDR, a B12.2 VH CDR, a1B18 VH CDR, a 2B18.1 VH CDR, a 4B19 VH CDR, and a 1B22 VH CDR; and/orat least one VL complementarity determining region selected from thegroup consisting of a 2A10 VL CDR, a 3E1VL CDR, a 3E2VL CDR, a 3E3VLCDR, a 3E4VL CDR, a 3E4.1VL CDR, a 3E5VL CDR, a 3E6VL CDR, a 3E6.1VLCDR, a 4E11VL CDR, a 4E13VL CDR, a 4E16VL CDR, a 4E16.1VL CDR, a 4E17VLCDR, a 4E17.1VL CDR, an A12 VL CDR, a 6A12 VL CDR, a B1.1 VL CDR, a B6VL CDR, a B6.1 VL CDR, a B8 VL CDR, a B8.1 VL CDR, a B11 VL CDR, a B11C3VL CDR, a B11E8 VL CDR, a B12 VL CDR, a B12.1. VL CDR, a B12.2 VL CDR, a1B18 VL CDR, a 2B18.1 VL CDR, a 4B19 VL CDR, and a 1B22 VL CDR. Invarious embodiments the antibody comprises the VH CDRs of an antibodyselected from the group consisting of 2A10, 3E1VH CDR, 3E2VH CDR, 3E3VHCDR, 3E4VH CDR, 3E4.1VH CDR, 3E5VH CDR, 3E6VH CDR, 3E6.1VH CDR, 4E11VHCDR, 4E13VH CDR, 4E16VH CDR, 4E16.1VH CDR, 4E17VH CDR, 4E17.1VH CDR,A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1,B12.2, 1818, 2818.1, 4819, and 1822; and/or the VL CDRs of an antibodyselected from the group consisting of 2A10, 3E1VH CDR, 3E2VH CDR, 3E3VHCDR, 3E4VH CDR, 3E4.1VH CDR, 3E5VH CDR, 3E6VH CDR, 3E6.1VH CDR, 4E11VHCDR, 4E13VH CDR, 4E16VH CDR, 4E16.1VH CDR, 4E17VH CDR, 4E17.1VH CDR,A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1,B12.2, 1818, 2B18.1, 4B19, and 1B22. In various embodiments the antibodycomprises the VH and VL CDRs of an antibody selected from the groupconsisting of 2A10, 3E1VH CDR, 3E2VH CDR, 3E3VH CDR, 3E4VH CDR, 3E4.1VHCDR, 3E5VH CDR, 3E6VH CDR, 3E6.1VH CDR, 4E11VH CDR, 4E13VH CDR, 4E16VHCDR, 4E16.1VH CDR, 4E17VH CDR, 4E17.1VH CDR, A12, 6A12, B1.1, B6, B6.1,B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2, 1818, 2B18.1, 4B1319,and 1B22. In various embodiments the antibody comprises the VH and VLdomains of an antibody selected from the group consisting of 2A10, 3E1VHCDR, 3E2VH CDR, 3E3VH CDR, 3E4VH CDR, 3E4.1VH CDR, 3E5VH CDR, 3E6VH CDR,3E6.1VH CDR, 4E11VH CDR, 4E13VH CDR, 4E16VH CDR, 4E16.1VH CDR, 4E17VHCDR, 4E17.1VH CDR, A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3,B11E8, B12, B12.1, B12.2, 1818, 2818.1, 4819, and 1B22. In certainembodiments the antibody is a single chain Fv (scFv), a FAB, a (Fab′)₂,an (ScFv)₂, and the like. In certain embodiments the antibody is an IgG.In certain embodiments the antibody is selected from the groupconsisting of 2A10, 3E1VH CDR, 3E2VH CDR, 3E3VH CDR, 3E4VH CDR, 3E4.1VHCDR, 3E5VH CDR, 3E6VH CDR, 3E6.1VH CDR, 4E11VH CDR, 4E13VH CDR, 4E16VHCDR, 4E16.1VH CDR, 4E17VH CDR, 4E17.1VH CDR, A12, 6A12, B1.1, B6, B6.1,B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2, 1B18, 2B18.1, 4B19, and1B22. In various embodiments the antibody is in a pharmaceuticallyacceptable excipient (e.g., in a unit dosage formulation).

In various embodiments method of method of inhibiting the activity ofBotulinum neurotoxin in a mammal are provided. The methods typicallyinvolve administering to a mammal in need thereof a compositioncomprising at least one neutralizing anti-BoNT antibody as describedherein. In certain embodiments the composition comprises at least twodifferent antibodies that each bind different BoNT serotypes. In certainembodiments the composition comprises at least three differentantibodies that each bind different BoNT epitopes.

In certain embodiments compositions are provided that partially or fullyneutralize a Botulinum neurotoxin (BoNT). The compositions typicallycomprise a first antibody that binds a BoNT/B or a BoNT/E serotype,e.g., one or more antibodies as described above, and a second antibodythat binds a BoNT serotype selected from the group consisting of BoNT/A,BoNT/B, BoNT/C, BoNT/D, BoNT/E, and BoNT/F.

In various embodiments nucleic acids are provided that encode one ormore antibodies as described herein. In certain embodiments cellscontaining such antibodies are also provided herein. Kits are alsoprovided for neutralizing a Botulinum neurotoxin. The kits typicallycomprise a composition comprising one or more antibodies as describedherein. The kits optionally also include instructional materialsteaching the use of the composition to neutralize a Botulinumneurotoxin. In certain embodiments the composition is stored in adisposable syringe.

DEFINITIONS

A “BoNT polypeptide” refers to a Botulinum neurotoxin polypeptide (e.g.,a BoNT/A polypeptide, a BoNT/B polypeptide, a BoNT/C polypeptide, and soforth). The BoNT polypeptide can refer to a full-length polypeptide orto a fragment thereof. Thus, for example, the term “BoNT/A polypeptide”refers to either a full-length BoNT/A (a neurotoxin produced byClostridium botulinum of the type A serotype) or a fragment thereof(e.g. the Hc fragment). The H_(C) fragment approximately a 50 DaC-terminal fragment (residues 873-1296) of BoNT/A (Lacy and Stevens(1999) J. Mol. Biol., 291: 1091-1104).

A “BoNT” serotype refers one of the standard known BoNT serotypes (e.g.BoNT/A, BoNT/C, BoNT/D, BoNT/E, BoNT/F, etc.). BoNT serotypes differfrom each other by as little as about 35% at the amino acid level (e.g.,between BoNT/E and BoNT/F) up to about 66% at the amino acid level,(e.g., for BoNT/A vs BoNT/C or D). Thus, BoNT serotypes differ from eachother by about 35-66% at the amino acid level.

The term “BoNT subtype” (e.g., a BoNT/A1A subtype) refers to botulinumneurotoxin gene sequences of a particular serotype (e.g., A, C, D, F,etc.) that differ from each other sufficiently to produce differentialantibody binding. In certain embodiments, the subtypes differ from eachother by at least 2.5%, preferably by at least 5%, or 10%, morepreferably by at least 15% or 20% at the amino acid level. In certainembodiments, the subtypes differ from each other by nor more than 35%,preferably by no more than 31.6%, still more preferably by no more than30%, or 25%, more preferably by less than about 20% or 16% at the aminoacid level. In certain embodiments, BoNT subtypes differ from each otherby at least 2.6%, more preferably by at least 3%, and most preferably byat least 3.6% at the amino acid level. BoNT subtypes typically differfrom each other by less than about 31.6%, more preferably by less thanabout 16%, at the amino acid level.

An “anti-BoNT antibody” refers to an antibody that binds a BoNTpolypeptide, preferably specifically binds a BoNT polypeptide with a KDless than 10⁻⁷, preferably less than 10⁻⁸, or 10⁻⁹, more preferably lessthan 10⁻¹⁰, 10⁻¹¹, or 10⁻¹².

“Neutralization” refers to a measurable decrease in the toxicity of aBotulinum neurotoxin (e.g., BoNT/A).

The term “high affinity” when used with respect to an antibody refers toan antibody that specifically binds to its target(s) with an affinity(K_(D)) of at least about 10⁻⁸ M, preferably at least about 10⁻⁹M, morepreferably at least about 10⁻¹⁰M, and most preferably at last about10⁻¹¹ M. In certain embodiments “high affinity” antibodies have a K_(D)that ranges from about 1 nM to about 5 pM.

The following abbreviations are used herein: AMP, ampicillin; BIG,botulinum immune globulin; BoNT, botulinum neurotoxin; BoNT/A, BoNT typeA; CDR, complementarity determining region; ELISA, enzyme-linkedimmunosorbent assay; GLU, glucose; HBS, HEPES-buffered saline (10 mMHEPES, 150 mM NaCl [pH 7.4]); H_(c), c-terminal domain of BoNT heavychain (binding domain); H_(N), N-terminal domain of BoNT heavy chain(translocation domain); IgG, immunoglobutin G; IMAC, immobilized-metalaffinity chromatography; IPTG, isopropyl-β-D-thiogalactopyranoside; KAN,kanamycin; K_(d), equilibrium constant; k_(off), dissociation rateconstant; k_(on), association rate constant; MPBS, skim milk powder inPBS; NTA, nitrilotriacetic acid; PBS, phosphate-buffered saline (25 mMNaH₂PO₄, 125 mM NaCl [pH 7.0]; RU, resonance units; scFv, single-chainFv antibody fragments; TPBS, 0.05% (vol/vol) Tween 20 in PBS; TMPBS,0.05% (vol/vol) Tween 20 in MPBS; TU, transducing units; V_(H),immunoglobulin heavy-chain variable region; V_(K), immunoglobulin kappalight-chain variable region; V_(L) immunoglobulin light-chain variableregion; wt, wild type.

The terms “polypeptide”, “peptide”, or “protein” are usedinterchangeably herein to designate a linear series of amino acidresidues connected one to the other by peptide bonds between thealpha-amino and carboxy groups of adjacent residues. The amino acidresidues are preferably in the natural “L” isomeric form. However,residues in the “D” isomeric form can be substituted for any L-aminoacid residue, as long as the desired functional property is retained bythe polypeptide. In addition, the amino acids, in addition to the 20“standard” amino acids, include modified and unusual amino acids, whichinclude, but are not limited to those listed in 37 CFR (1.822(b)(4).Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates either a peptide bond to afurther sequence of one or more amino acid residues or a covalent bondto a carboxyl or hydroxyl end group.

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include, but are notlimited to, Fab′₂, IgG, IgM, IgA, and single chain antibodies, morepreferably single chain Fv (scFv) antibodies in which a variable heavyand a variable light chain are joined together (directly or through apeptide linker) to form a continuous polypeptide.

An “antigen-binding site” or “binding portion” refers to the part of animmunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions”or “FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen binding “surface”. This surface mediates recognition andbinding of the target antigen. The three hypervariable regions of eachof the heavy and light chains are referred to as “complementaritydetermining regions” or “CDRs” and are characterized, for example byKabat et al. Sequences of proteins of immunological interest, 4th ed.U.S. Dept. Health and Human Services, Public Health Services, Bethesda,Md. (1987).

An S25 antibody refers to an antibody expressed by clone S25 or to anantibody synthesized in other manners, but having the same CDRs andpreferably, but not necessarily, the same framework regions as theantibody expressed by clone s25. Similarly, antibodies C25, 1C6, 3D12,B4, 1F3, HuC25, AR1, AR2, AR3, AR4, WR1(V), WR1(T), 3-1, 3-8, 3-10,ING1, CR1, RAZZ, or ING2 refer to antibodies expressed by thecorresponding clone(s) and/or to antibodies synthesized in othermanners, but having the same CDRs and preferably, but not necessarily,the same framework regions as the referenced antibodies.

As used herein, the terms “immunological binding” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smaller Kdrepresents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and on geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.The ratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity and is thus equal to the dissociation constant K_(d)(see, generally, Davies et al. (1990) Ann. Rev. Biochem., 59: 439-473).

A “BoNT-neutralizing antibody” refers to an antibody that binds to oneor more Botulinum neurotoxin(s) (e.g., BoNT/A1, BoNT/A2, etc.) and thatby so-binding reduces the toxicity of that BoNT neurotoxin. Thus, forexample the term “BoNT/A-neutralizing antibody”, as used herein refersto an antibody that specifically binds to a BoNT/A polypeptide (e.g. aBoNT/A1 polypeptide), in certain embodiments, to an H_(C) domain of aBoNT/A polypeptide and that by so-binding reduces the toxicity of theBoNT/A polypeptide. Reduced toxicity can be measured as an increase inthe time that paralysis developed and/or as a lethal dosage (e.g., LD₅₀)as described herein. Antibodies derived from BoNT-neutralizingantibodies include, but are not limited to, the antibodies whosesequence is expressly provided herein.

Antibodies derived from BoNT-neutralizing antibodies preferably have abinding affinity of about 1.6×10⁻⁸ or better and can be derived byscreening libraries of single chain Fv fragments displayed on phage oryeast constructed from heavy (VH) and light (VL) chain variable regiongenes obtained from mammals, including mice and humans, immunized withbotulinum toxoid, toxin, or BoNT fragments. Antibodies can also bederived by screening phage or yeast display libraries in which a knownBoNT-neutralizing variable heavy (V_(H)) chain is expressed incombination with a multiplicity of variable light (V_(L)) chains orconversely a known BoNT-neutralizing variable light chain is expressedin combination with a multiplicity of variable heavy (V_(H)) chains.BoNT-neutralizing antibodies also include those antibodies produced bythe introduction of mutations into the variable heavy or variable lightcomplementarity determining regions (CDR1, CDR2 or CDR3) as describedherein. Finally BoNT-neutralizing antibodies include those antibodiesproduced by any combination of these modification methods as applied tothe BoNT-neutralizing antibodies described herein and their derivatives.

A neutralizing epitope refers to the epitope specifically bound by aneutralizing antibody.

A single chain Fv (“scFv” or “scFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which may be expressed from a nucleic acidincluding V_(H)- and V_(L)-encoding sequences either joined directly orjoined by a peptide-encoding linker. Huston, et al. (1988) Proc. Nat.Acad. Sci. USA, 85: 5879-5883. A number of structures for converting thenaturally aggregated—but chemically separated light and heavypolypeptide chains from an antibody V region into an scFv molecule whichwill fold into a three dimensional structure substantially similar tothe structure of an antigen-binding site. See, e.g. U.S. Pat. Nos.5,091,513 and 5,132,405 and 4,956,778.

In one class of embodiments, recombinant design methods can be used todevelop suitable chemical structures (linkers) for converting twonaturally associated—but chemically separate—heavy and light polypeptidechains from an antibody variable region into a scFv molecule which willfold into a three-dimensional structure that is substantially similar tonative antibody structure.

Design criteria include determination of the appropriate length to spanthe distance between the C-terminal of one chain and the N-terminal ofthe other, wherein the linker is generally formed from small hydrophilicamino acid residues that do not tend to coil or form secondarystructures. Such methods have been described in the art. See, e.g., U.S.Pat. Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Pat. No.4,946,778 to Ladner et al.

In this regard, the first general step of linker design involvesidentification of plausible sites to be linked. Appropriate linkagesites on each of the V_(H) and V_(L) polypeptide domains include thosewhich will result in the minimum loss of residues from the polypeptidedomains, and which will necessitate a linker comprising a minimum numberof residues consistent with the need for molecule stability. A pair ofsites defines a “gap” to be linked. Linkers connecting the C-terminus ofone domain to the N-terminus of the next generally comprise hydrophilicamino acids which assume an unstructured configuration in physiologicalsolutions and preferably are free of residues having large side groupswhich might interfere with proper folding of the V_(H) and V_(L) chains.Thus, suitable linkers under the invention generally comprisepolypeptide chains of alternating sets of glycine and serine residues,and may include glutamic acid and lysine residues inserted to enhancesolubility. One particular linker under the invention has the amino acidsequence [(Gly)₄Ser]₃ (SEQ ID NO:1). Another particularly preferredlinker has the amino acid sequence comprising 2 or 3 repeats of[(Ser)₄Gly] (SEQ ID NO:2), such as [(Ser)₄Gly]₃ (SEQ ID NO:3), and thelike. Nucleotide sequences encoding such linker moieties can be readilyprovided using various oligonucleotide synthesis techniques known in theart (see, e.g., Sambrook, supra.).

The phrase “specifically binds to a protein” or “specificallyimmunoreactive with”, when referring to an antibody refers to a bindingreaction which is determinative of the presence of the protein in thepresence of a heterogeneous population of proteins and other biologics.Thus, under designated immunoassay conditions, the specified antibodiesbind to a particular protein and do not bind in a significant amount toother proteins present in the sample. Specific binding to a proteinunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, BoNT/A-neutralizingantibodies can be raised to BoNT/A protein(s) that specifically bind toBoNT/A protein(s), and not to other proteins present in a tissue sample.A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select monoclonalantibodies specifically immunoreactive with a protein. See Harlow andLane (1988) Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, for a description of immunoassay formats andconditions that can be used to determine specific immunoreactivity.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Typically conservative amino acid substitutions involve substitution oneamino acid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another: 1)Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates one strategy for in vitro antibody production usingphage libraries. mRNA is prepared from splenocytes, first-strand cDNA isprepared, and antibody V_(H) and V_(L) genes are amplified by PCR. V_(H)and V_(L) genes are spliced together randomly using PCR to create arepertoire of scFv genes. The scFv gene repertoire is cloned into aphagemid vector in frame with a gene (gIII) encoding a phagemid minorcoat protein (pIII). Each phage in the resulting phage antibody libraryexpresses and scFv-pIII fusion protein on its surface and contains thegene encoding the scFv inside. Phage antibodies binding a specificantigen can be separated from nonbinding phage antibodies by affinitychromatography on immobilized antigen. A single round of selectionincreases the number of antigen-binding phage antibodies by a factorranging from 20 to 10,000 depending on the affinity of the antibody.Eluted phage antibodies are used to infect E. coli, which then producemore phage antibodies for the next round of selection. Repeated roundsof selection make it possible to isolate antigen-binding phageantibodies that were originally present at frequencies of less than onein a billion.

FIG. 2 shows deduced protein sequences of heavy (VH) and light (VL)chain variable regions of BoNT B binders. VH domains: A12 (SEQ ID NO:4),6A12 (SEQ ID NO:5), B1.1 (SEQ ID NO:6), B6 (SEQ ID NO:7), B6.1 (SEQ IDNO:8), B8 (SEQ ID NO:9), B8.1 (SEQ ID NO:10), B11 (SEQ ID NO:11), B11C3(SEQ ID NO:12), B11E8 (SEQ ID NO:13), B12 (SEQ ID NO:14), B12.1 (SEQ IDNO:15), B12.2 (SEQ ID NO:16), 1B18 (SEQ ID NO:17), 2B18.1 (SEQ IDNO:18), 41319 (SEQ ID NO:19), 1B22 (SEQ ID NO:20). VL domains: A12 (SEQID NO:21), 6A12 (SEQ ID NO:22), B1.1 (SEQ ID NO:23), B6 (SEQ ID NO:24),B6.1 (SEQ ID NO:25), B8 (SEQ ID NO:26), B8.1 (SEQ ID NO:27), B11 (SEQ IDNO:28), B11C3 (SEQ ID NO:29), B11E8 (SEQ ID NO:30), B12 (SEQ ID NO:31),B12.1 (SEQ ID NO:32), B12.2 (SEQ ID NO:33), 1B18 (SEQ ID NO:34), 2B18.1(SEQ ID NO:35), 4B19 (SEQ ID NO:36), 1B22 (SEQ ID NO:37). Dashesindicate conserved residues. Letters indicate mutated residues.

FIG. 3 shows deduced protein sequences of heavy and light chain variableregions of BoNT/E binders. VH domains: 2A10 (SEQ ID NO:38), 3E1 (SEQ IDNO:39), 3E2 (SEQ ID NO:40), 3E3 (SEQ ID NO:41), 3E4 (SEQ ID NO:42),3E4.1 (SEQ ID NO:43), 3E5 (SEQ ID NO:44), 3E6 (SEQ ID NO:45), 3E6.1 (SEQID NO:46), 4E11 (SEQ ID NO:47), 4E13 (SEQ ID NO:48), 4E16 (SEQ IDNO:49), 4E16.1 (SEQ ID NO:50), 4E17 (SEQ ID NO:51), 4E17.1 (SEQ IDNO:52); VL domains: 2A10 (SEQ ID NO:53), 3E1 (SEQ ID NO:54), 3E2 (SEQ IDNO:55), 3E3 (SEQ ID NO:56), 3E4 (SEQ ID NO:57), 3E4.1 (SEQ ID NO:58),3E5 (SEQ ID NO:59), 3E6 (SEQ ID NO:60), 3E6.1 (SEQ ID NO:61), 4E11 (SEQID NO:62), 4E13 (SEQ ID NO:63), 4E16 (SEQ ID NO:64), 4E16.1 (SEQ IDNO:65), 4E17 (SEQ ID NO:66), 4E17.1 (SEQ ID NO:67) Dashes indicateconserved residues. Letters indicate mutated residues.

FIG. 4 shows a phylogenetic tree of published botulinum neurotoxingenes. The phylogenetic tree was constructed from the DNA sequences ofpublished Clostridial neurotoxin genes using Vector NTI software.

FIG. 5 shows an analysis of BoNT/A gene sequences. The phylogenetic treeof BoNT/A genes reveals two clusters, A1 and A2.

FIG. 6 shows an analysis of BoNT/B gene sequences. A phylogenetic treeof BoNT/B genes reveals four clusters: BoNT/B1, BoNT/B2, nonproteolyticBoNT/B, and bivalent BoNT/B. Percent differences between clusters rangefrom 3.6 to 7.7%. As with BoNT/A, the greatest differences are seen inthe heavy chain.

FIGS. 7A and 7B show a scheme used for affinity maturation of HuC25(FIG. 7A) and 3D12 (FIG. 7B) scFv using yeast display.

DETAILED DESCRIPTION

This invention provides novel antibodies that specifically bind to andneutralize botulinum neurotoxin type B and E, and in certainembodiments, other botulinum neurotoxin serotypes (e.g., A, C, D, F,etc., see, e.g., FIGS. 4-6). Botulinum neurotoxin is produced by theanaerobic bacterium Clostridium botulinum. Botulinum neurotoxinpoisoning (botulism) arises in a number of contexts including, but notlimited to food poisoning (food borne botulism), infected wounds (woundbotulism), “infant botulism” from ingestion of spores and production oftoxin in the intestine of infants, and as aa chemical/biological warfareagent. Botulism is a paralytic disease that typically begins withcranial nerve involvement and progresses caudally to involve theextremities. In acute cases, botulism can prove fatal.

Botulism neurotoxins (BoNTs) are classified by the Centers for DiseaseControl (CDC) as one of the six highest-risk threat agents forbioterrorism (the “Category A agents”), due to their extreme potency andlethality, ease of production and transport, and the need for prolongedintensive care (Amon et al. (2001) JAMA 285: 1059-1070). Both Iraq andthe former Soviet Union produced BoNT for use as weapons (UN SecurityCouncil (1995) supra; Bozheyeva (1999) supra.) and the Japanese cult AumShinrikyo attempted to use BoNT for bioterrorism (Arnon (2001) supra.).As a result of these threats, specific pharmaceutical agents are neededfor prevention and treatment of intoxication.

It has recently been discovered that there are multiple subtypes ofvarious BoNT serotypes. Moreover, we have further discovered that manyantibodies that bind, for example the BoNT/A1 subtype will not bind theBoNT/A2 subtype, and so forth

In certain embodiments this invention pertains to the discovery thatthat particularly efficient neutralization of a botulism neurotoxin(BoNT) subtype is achieved by the use of neutralizing antibodies thatbind two or more subtypes of the particular BoNT serotype with highaffinity. In various embodiments this can be accomplished by using twoor more different antibodies directed against each of the subtypes, oralternatively, by the use of antibodies that are cross-reactive fordifferent BoNT subtypes, or by bispecific or polyspecific antibodieswith specificities for two or more BoNT epitopes, and/or serotypes,and/or subtypes.

It was also a surprising discovery that when one starts combiningneutralizing antibodies that the potency of the antibody combinationincreases dramatically. This increase makes it possible to generate amulti-antibody, and/or multi-specific antibodies of the required potencyfor therapeutic use. It was also surprising that as one begins combiningtwo and three monoclonal antibodies, the particular BoNT epitope that isrecognized becomes less important. Thus, in certain embodiments, thisinvention contemplates compositions comprising at least two, morepreferably at least three high affinity antibodies that bindnon-overlapping epitopes on the BoNT.

Thus, in certain embodiments, this invention contemplates compositionscomprising two or more, in certain embodiments preferably three or moredifferent antibodies selected from the antibodies described herein (see,e.g., FIGS. 2, and 3) and/or antibodies comprising one or more CDRs fromthese antibodies, and/or one or more antibodies comprising mutants ofthese antibodies.

As indicated above, in certain embodiments, the antibodies provided bythis invention bind to and neutralize one or more botulinum neurotoxintype B, E, and in certain instances Bont/A subtypes. Neutralization, inthis context, refers to a measurable decrease in the toxicity of thetarget neurotoxin. Such a decrease in toxicity can be measured in vitroby a number of methods well known to those of skill in the art. One suchassay involves measuring the time to a given percentage (e.g., 50%)twitch tension reduction in a hemidiaphragm preparation. Toxicity can bedetermined in vivo, e.g. as an LD₅₀ in a test animal (e.g. mouse)botulinum neurotoxin type A in the presence of one or more putativeneutralizing antibodies. The neutralizing antibody or antibodycombination can be combined with the botulinum neurotoxin prior toadministration, or the animal can be administered the antibody prior to,simultaneous with, or after administration of the neurotoxin.

As the antibodies of this invention act to neutralize botulinumneurotoxins, they are useful in the treatment of pathologies associatedwith botulinum neurotoxin poisoning. The treatments essentially compriseadministering to the poisoned organism (e.g. human or non-human mammal)a quantity of one or more neutralizing antibodies sufficient toneutralize (e.g. mitigate or eliminate) symptoms of BoNT poisoning.

Such treatments are most desired and efficacious in acute cases (e.g.where vital capacity is less than 30-40 percent of predicted and/orparalysis is progressing rapidly and/or hypoxemia with absolute orrelative hypercarbia is present. These antibodies can also be used totreat early cases with symptoms milder than indicated (to preventprogression) or even prophylactically (a use the military envisions forsoldiers going in harms way). Treatment with the neutralizing antibodycan be provided as an adjunct to other therapies (e.g. antibiotictreatment).

The antibodies provided by this invention can also be used for the rapiddetection/diagnosis of botulism (type B, E, or A toxin(s)) and therebysupplement and/or replace previous laboratory diagnostics.

In another embodiment this invention provides the epitopes specificallybound by botulinum neurotoxin antibodies described herein. Theseepitopes can be used to isolate, and/or identify and/or screen for otherantibodies BoNT neutralizing antibodies as described herein.

I. Potency of Botulinum Neurotoxin (BoNT)-Neutralizing Antibodies.

Without being bound to a particular theory, it is believed that thecurrent antitoxins used to treat botulism (horse and human) have apotency of about 5000 mouse LD50s/mg (human) and 55,000 mouse LD50s mg(horse).

Based on our calculations, we believe a commercially desirable antitoxinwill have a have a potency greater than about 10,000 to 100,000LD50s/mg. Combinations of the antibodies described herein (e.g., two orthree antibodies) can meet this potency. Thus, in certain embodiments,this invention provides antibodies and/or antibody combinations thatneutralize at least about 10,000 mouse LD50s/mg of antibody, preferablyat least about 15,000 mouse LD50s/mg of antibody, more preferably atleast about 20,000 mouse LD50s/mg of antibody, and most preferably atleast about 25,000 mouse LD50s/mg of antibody.

II. Botulinum Neurotoxin (BoNT)-Neutralizing Antibodies.

In certain preferred embodiments, BoNT neutralizing antibodies areselected that bind to or more BoNT subtypes. A number of subtypes areknown for each BoNT serotype. Thus, for example, BoNT/A subtypesinclude, but are not limited to, BoNT/A1, BoNT/A2, BoNT/A3, and the like(see, e.g., FIG. 4). It is also noted, for example, that the BoNT/A1subtype includes, but is not limited to 62A, NCTC 2916, ATCC 3502, andHall hyper (Hall Allergan) and are identical (99.9-100% identity at theamino acid level.) and have been classified as subtype A1 (FIG. 5A). TheBoNT/A2 sequences (Kyoto-F and FRI-A2H) (Willems, et al. (1993) Res.Microbiol. 144:547-556) are 100% identical at the amino acid level.Another BoNT/A subtype, (that we are calling A3) is produced by a straincalled Loch Maree that killed a number of people in an outbreak inScotland.

Similarly, as shown in FIG. 4, a number of subtypes are also known forserotypes B, C, E, and F. Using, the methods described herein, it wasdiscovered that high-affinity antibodies that are cross-reactive withtwo or more subtypes within a serotype can also be produced (e.g.,selected/engineered). Moreover, without being bound to a particulartheory, it appears that these cross-reactive antibodies cansubstantially more efficient in neutralizing Botulinum neurotoxin,particularly when used in combination one or more different neutralizingantibodies.

The sequences of the variable heavy (VH) and variable light (VL) domainsfor a number of prototypical BoNT/B and BoNT/E antibodies areillustrated in Tables 1-4, and in FIGS. 2-3.

These antibodies can be used individually, and/or in combination witheach other, and/or in combination with other known anti-BoNT antibodies(see, e.g., copending application Ser. No. 11/342,27, filed on Jan. 26,2006, Ser. No. 09/144,886, filed in Aug. 31, 1998, Ser. No. 10/632,706,filed on Aug. 1, 2003, and PCT application Nos: PCT/US2006/003070 andPCT/US03/24371, which are incorporated herein by reference for allpurposes) to form bispecific or polyspecific antibodies

TABLE 1 Deduced protein sequences of heavy chain variableregions of BoNT/E binders. VH Clone/ Gene Framework Framework FrameworkFramework Family 1 CDR1 2 CDR2 3 CDR3 4 2A10 QVQLQQS RYTIT WVRQAPGGIIPIFDKA RVTFTAD YSRGY WGPGTL VH1 GAEVKKP (SEQ ID QGLEWM NYAQKFQASTSTAY VHFDY VTVSS GSSVKVS NO: 69) G S MELGSLR (SEQ ID (SEQ ID CKASGGT(SEQ ID (SEQ ID PEDTAVY NO: 73) NO: 74) FT NO: 70) NO: 71) YCAA (SEQ ID(SEQ ID NO: 68) NO: 72) 3E1 QVQLVES NSGFT WVRQVPG GIIPMFGP RVTITADEDQGEY WGEGTT VH1 GAEVKKP (SEQ ID QGLEWM ANYAQKF STRMVYM TVGML VTVSSGSSVKVS NO: 76) G QG ELRSLRSE LYYAM (SEQ ID CKASGGT (SEQ ID (SEQ IDDTAVYYC DV NO: 81) FS NO: 77) NO: 78) AR (SEQ ID (SEQ ID (SEQ ID NO: 80)NO: 75) NO: 79) 3E2 QVQLQES KYAIT WLRQAPG GITPIFATT RVMITAD SPRGGIWGQGTM VH1 GAEVKKP (SEQ ID QGFEWMG NYAQKFQ EVTSTVY VGTFD VTVSS GSSVKVSNO: 83) (SEQ ID G MDLSSLG T (SEQ ID CKASGGD NO: 84) (SEQ ID SEDTAIYF(SEQ ID NO: 88) LN NO: 85) CAK NO: 87) (SEQ ID (SEQ ID NO: 82) NO: 86)3E3 QVQLVES NYNMN WVRQAPG SISDGGSY RFTISRDN DEMVH WGQGTT VH3 GGGLVKP(SEQ ID KGLEWVS RYYAYSV TKNSLYL GILVYY VTVSS GESLRLSC NO: 90) (SEQ ID KGQMNSLRA GMDV (SEQ ID AASGFTFS NO: 91) (SEQ ID EDTALYY (SEQ ID NO: 95)(SEQ ID NO: 92) CAR NO: 94) NO: 89) (SEQ ID NO: 93) 3E4 QVQLQES SDAMSWVRQAPG AILPSGEA RFTISRHS DSYHS WGQGTM VH3 GGGLVQP (SEQ ID KGLEWVATYYADSV SKNTLYL RLAAF VTVSS GGSLRLSC NO: 97) (SEQ ID KG QMNSLRA DI(SEQ ID GASGFTFS NO: 98) (SEQ ID DDTAVYY (SEQ ID NO: 102) (SEQ IDNO: 99) CAR NO: 101) NO: 96) (SEQ ID NO: 100) 3E4.1 QVQLQES SDAMSWVRQAPG AILPSGEA RFTISRHS DSYHS WGQGTM VH3 GGGLVQP (SEQ ID KGLEWVATYYADSV SKNTLYL RLAAF VTVSS GGSLRLSC NO: 104) (SEQ ID KG QMNSLRA DI(SEQ ID GASGFTFS NO: 105) (SEQ ID DDTAVYY (SEQ ID NO: 109) (SEQ IDNO: 106) CAR NO: 108) NO: 103) (SEQ ID NO: 107) 3E5 QVQLVQS DFYMSWIRQAPG YIGSSGSA RFTISRDN VASRY WGQGTM VH3 GGGVVQP (SEQ ID KGLEWVSLQYADSV DKNVLYL HDVLT VTVSS GRPLRLSC NO: 111) (SEQ ID KG QMTSLRA DGFDI(SEQ ID AASTFNFR NO: 112) (SEQ ID EDTAVYY (SEQ ID NO: 116) (SEQ IDNO: 113) CAR NO: 115) NO: 110) (SEQ ID NO: 114) 3E6 QVQLVQS SYAMHWVRQAPG VISYDGN RFTISRDN ARLCTS WGQGTL VH3 GGGVVQP (SEQ ID KGLEWVAKKYYADS SKNTLYL TSCYW VTVSS GKSLRLSC NO: 118) (SEQ ID VKG QMNSLRA TFDP(SEQ ID AASGFTFS NO: 119) (SEQ ID EDAAVFY (SEQ ID NO: 123) (SEQ IDNO: 120) CAR NO: 122) NO: 117) (SEQ ID NO: 121) 3E6.1 QVQLVQS SYAMHWVRQAPG VISYDGN RFTISRDN ARLCTS WGQGTL VH3 GGGVVQP (SEQ ID KGLEWVAKKYYADS SKNTLYL TSCYW VTVSS GKSLRLSC NO: 125) (SEQ ID VKG QMNSLRA TFDP(SEQ ID AASGFTFS NO: 126) (SEQ ID EDAAVFY (SEQ ID NO: 130) (SEQ IDNO: 127) CAR NO: 129) NO: 124) (SEQ ID NO: 128) 4E11 QVQLVQS GYSFNWVRQAPG YMSSGGSI RFTISRDN GPPGRP WGQGTM VH3 GGGLVQP (SEQ ID KGLEWVAKNYADSV AKNSLYL NDAFDI VTVSS GGSLRLSC NO: 132) (SEQ ID KG QVNSLRD(SEQ ID (SEQ ID AASGFRFS NO: 133) (SEQ ID EDTALYY NO: 136) NO: 137)(SEQ ID NO: 134) CAR NO: 131) (SEQ ID NO: 135) 4E13 EVQLVQS SYAMTWVRQAPG SISVSGDS RFTISRDN GLSKA WGQGTM VH3 GGGLVQP (SEQ ID KGLEWVSTYYADSV SKNTVSL DLFGM VTVSS GGSLRLSC NO: 139) (SEQ ID KG QMNSLRA DV(SEQ ID AASGFTFS NO: 140) (SEQ ID EDTALYY (SEQ ID NO: 144) (SEQ IDNO: 141) CAK NO: 143) NO: 138) (SEQ ID NO: 142) 4E16 QVQLQES DYYWSWIRQPPG YIYYSGST RVTISVDT HTSGW WGQGTM VH4 GPGLVKPS (SEQ ID KGLEWIGNYNPSLKS SKNQFSLN SGGAF VTVSS ETLSLTCS NO: 145) (SEQ ID (SEQ ID LSSVTAADI (SEQ ID VSGVSIS NO: 146) NO: 147) DTAVYYC (SEQ ID NO: 150) ARNO: 149) (SEQ ID NO: 148) 4E16.1 QVQLQES DYYWS WIRQPPG YIYYSGST RVTISVDTHTSGW WGQGTM VH4 GPGLVKPS (SEQ ID KGLEWIG NYNPSLKS SKNQFSLN SGGAF VTVSSETLSLTCS NO: 152) (SEQ ID (SEQ ID LSSVTAA DI (SEQ ID VSGVSIS NO: 153)NO: 154) DTAVYYC (SEQ ID NO: 157) (SEQ ID AR NO: 156) NO: 151) (SEQ IDNO: 155) 4E17 EVQLVQS HWMT WVRQAPG NINLDGTE RFTVSRD LQWGG WGQGTL VH3GGNLVQP (SEQ ID QGLEWVA KFYVDSV NRKSSVFL YNGWL VTVSS GGSLRLSC NO: 159)(SEQ ID KG QMNNLRV SP (SEQ ID AATGPIGS NO: 160) (SEQ ID DDTAVYY (SEQ IDNO: 164) (SEQ ID NO: 161) CAR NO: 163) NO: 158) (SEQ ID NO: 162) 4E17.1EVQLVQS HWMT WVRQAPG NINLDGTE RFTVSRD LQWGG WGQGTL GGNLVQP (SEQ IDQGLEWVA KFYVDSV NRKSSVFL YNGWL VTVSS GGSLRLSC NO: 166) (SEQ ID KGQMNNLRV SP (SEQ ID AATGPIGS NO: 167) (SEQ ID DDTAVYY (SEQ ID NO: 171)(SEQ ID NO: 168) CAR NO: 170) NO: 165) (SEQ ID NO: 169)

TABLE 2 Deduced protein sequences of light chain variable regions(VL) of BoNT/E binders. VL Clone/ Gene Framework Framework FrameworkFramework Family 1 CDR1 2 CDR2 3 CDR3 4 E1 DIVMTQSP WASQG WYQQKPG AASTLQGVPSRFSGS QQLNSY FGGGTK VK1 SFLSASVG ISSYLA KAPKLLIY S (SEQ GSGTEFTLTIPLT (SEQ VDIKR (ZA1D DRVTITC (SEQ ID (SEQ ID ID SSLQPEDFA ID (SEQ IDVK1) (SEQ ID NO: 173) NO: 174) NO: 175) TYYC (SEQ NO: 177) NO: 178)NO: 172) ID NO: 176) 3E1 EIVLTQSP RASQGI WYQHKA AASSLQ GVPSRFSGS QQYNSYFGGGTK VK1 DSLSASVG SGYLA GKAPKLLI S (SEQ GYGTEFTLTI PFT (SEQ VEIKRDRVTITC (SEQ ID Y (SEQ ID ID SSLQPDDFA ID (SEQ ID (SEQ ID NO: 180)NO: 181) NO: 182) TYYC (SEQ NO: 184) NO: 185) NO: 179) ID NO: 183) 3E2EIVLTQSP RTSQSI WYQQKA AASTLH GVPSRFSGS QQSYSIP FGGGTK VK1 SFLSAFVGNNYLN GKAPKLLI T (SEQ GSGTEFTLTI LT (SEQ VEIKR DRVTITC (SEQ ID Y (SEQ IDID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 187) NO: 188) NO: 189) TYYC (SEQNO: 191) NO: 192) NO: 186) ID NO: 190) 3E3 DIVMTQSP RASQSF WYQQKPGAASSRA GVPTGSVAD QQSYST FGGGTK VK3 DSLSASVG SSSYLA QAPRLLIY A (SEQGSGTGFTLTI PYT VEIKR DSVTITC (SEQ ID (SEQ ID ID SGLQPEDFA (SEQ ID(SEQ ID (SEQ ID NO: 194) NO: 195) NO: 196) AYYC (SEQ NO: 198) NO: 199)NO: 193) ID NO: 197) 3E4 DIVMTQSP RASQSI WYQQKPG KASSLE GVPSRFSGS QQYNAFGGGTK VK1 SFLSAFVG SNWLA KAPKVLIY N (SEQ GSGTDFTLTI YPLT VEIKR DRVTITC(SEQ ID (SEQ ID ID TSLQPDDFA (SEQ ID (SEQ ID (SEQ ID NO: 201) NO: 202)NO: 203) TYYC (SEQ NO: 205) NO: 206) NO: 200) ID NO: 204) 3E4.1 EIVLTQSPRASQRI WYQQKPG KAFSLE GVPSRFSGS QQYDSY FGQGTKL VK1 STLSASVG GSWLAKAPNPLIY S (SEQ RSGTEFTLTI PYT EIKR DRVAITC (SEQ ID (SEQ ID ID SSLQPDDFA(SEQ ID (SEQ ID (SEQ ID NO: 208) NO: 209) NO: 210) TYFC (SEQ NO: 212)NO: 213) NO: 207) ID NO: 211) EK5 DVVMTQS QASQDI WYQQKPG DASNLEGVPSRFSGS QQYDPL FGGGTK VK1 PSSLSASIG SNRLN KVPKLLIS T (SEQ GSGTDFTFTILT (SEQ VEIKR DRVTFTC (SEQ ID (SEQ ID ID SSLQPEDIAT ID (SEQ ID (SEQ IDNO: 215) NO: 216) NO: 217) YYC (SEQ ID NO: 219) NO: 220) NO: 214)NO: 218) 3E6 DIQMTQSP RASQGI WYQQKSG AASSLQ GVPSRFSGS QQAYRT FGGGTK VK1SSVSASVG SSWLA QAPTLLIY S (SEQ GSGTDFTLII PIT (SEQ VEIKR DTVTISC (SEQ ID(SEQ ID ID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 222) NO: 223) NO: 224)TYYC (SEQ NO: 226) NO: 227) NO: 221) ID NO: 225) 3E6.1 DIQMTQSP QASQDIWYQQKPG AASSLQ GVPSRFSGS QQSYNT FGQGTKL VK1 SSVSASVG SNYLN KAPKLLIYS (SEQ GSGTDFTLTI PPT (SEQ EIKR DRVSITC (SEQ ID (SEQ ID ID SSLQPEDFA ID(SEQ ID (SEQ ID NO: 229) NO: 230) NO: 231) TYYC (SEQ NO: 233) NO: 234)NO: 228) ID NO: 232) 4E11 ASVLTQD QGDSL WYQQKPG GKSNRP GIPDRFSGSS NSRDSTFGGGTK VL3 PAVSVAL RSYYA QAPVLVIY S (SEQ SGNTASLTTI GNQL VTVLG GQTVRITCS (SEQ (SEQ ID ID GAQAEDEA (SEQ ID (SEQ ID (SEQ ID ID NO: 237) NO: 238)DYYC (SEQ NO: 240) NO: 241) NO: 235) NO: 236) ID NO: 239) 4E13 AELTQDPQGDSL WYQQKPG GENSRP GIPDRFSGSS NSPDSS FGGGTK VL3 AVSVALG RSYYA QAPVLVIYS (SEQ SGNTASLTI GIHLV VTVLG QTVRITC S (SEQ (SEQ ID ID AGAQAEDE (SEQ ID(SEQ ID (SEQ ID ID NO: 244) NO: 245) ADYYC (SEQ NO: 247) NO: 248)NO: 242) NO: 243) ID NO: 246) 4E16 EIVLTQSP KSSQSV WYQQKPG WASTREGVPDRFSGS HQYYSS FGGGTKL VK4 DSLSVSL LYSSN QPPKLLFY S (SEQ GSGTDFTLTIPLY (SEQ EIKR GERATINC NKNYL (SEQ ID ID SSLQAEDVA ID (SEQ ID (SEQ IDA (SEQ NO: 251) NO: 252) VYYC (SEQ NO: 254) NO: 255) NO: 249) IDID NO: 253) NO: 250) 4E16.1 EIVLTQSP KSSQSV WYQQKPG WASTRE GVPDRFSGSQQYYSS FGQGTKL VK4 NSLAVSL LYSGN QPPKLLIY S (SEQ GSETDFTLTI RWT EIKRGERATIRC NKNYI (SEQ ID ID SSLRAEDVA (SEQ ID (SEQ ID (SEQ ID A (SEQNO: 258) NO: 259) LYYC (SEQ NO: 261) NO: 262) NO: 256) ID ID NO: 260)NO: 257) 4E17 DIVMTQSP RASQSI WYQQKPG GTSNLQ GVPSGFSGS QETYST FGGGTKLVK1 SSVSASVG SSYLN KAPKLLIY S (SEQ GSGTDFTLTI PPT (SEQ EIKR DRVTITC(SEQ ID (SEQ ID ID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 264) NO: 265)NO: 266) TYYC (SEQ NO: 268) NO: 269) NO: 263) ID NO: 267) 4E17.1DIVMTQSP RASQSI WYQQKPG KASSLA GAPSRFSGS QQSYSIP FGGGTK VK1 SSLSASVGRHYVN KAPKLLIY S (SEQ GSGTDFTLTI LT (SEQ VEIKR DRVTISC (SEQ ID (SEQ IDID SSLQPDDFA ID (SEQ ID (SEQ ID NO: 271) NO: 272) NO: 273) TYYC (SEQNO: 275) NO: 276) NO: 270) ID NO: 274)

TABLE 3 Deduced protein sequences of heavy chain variable regions ofBoNT/B binders. VH Clone/ Gene Framework Framework Framework FrameworkFamily 1 CDR1 2 CDR2 3 CDR3 4 A12 EVQLVES SYGMH WVRQAP VIWYD RFTISRDNSKGYSNYD WGQGTT VH3 GGGVVQP (SEQ ID GKGLEW GSNKY NTLYLQMN YYYGM VTVSSGRSLRLSC NO: 278) VA (SEQ YADSV SLRAEDTAV DV (SEQ (SEQ ID AASGFTFS ID KGYYCAR (SEQ ID NO: 283) (SEQ ID NO: 279) (SEQ ID ID NO: 281) NO: 282)NO: 277) NO: 280) 6A12 QVQLVES SYGMH WVRQAP YISSSG RFTISRDNA VSIVGGWGQGTT VH3 GGGVVQP (SEQ ID GKGLEW STIYYA KNSLYLQM PYGMD VTVSS GRSLRLSCNO: 285) VS (SEQ DSVKG NSLRAEDTA V (SEQ (SEQ ID AASGFTFS ID (SEQ IDVYYCAR ID NO: 290) (SEQ ID NO: 286) NO: 287) (SEQ ID NO: 289) NO: 284)NO: 288) B1.1 QVQLVQS SYAFT WVRQAP RIVPFL RVTITADKA DKRTYE WGRGTL VH1GAEVEKP (SEQ ID GQGLEW GVPYY TSTVYMELS YNWNSL VTVSS GSSVKVS NO: 292)MG (SEQ TQKFR SLTFDDTAV WF (SEQ (SEQ ID CKASGGS ID G (SEQ YYCAR (SEQ IDNO: 297) FS (SEQ ID NO: 293) ID ID NO: 295) NO: 296) NO: 291) NO: 294)B6 QVQLVQS SFWIA WVRQMP IIYAGD HVNISVDRS HDSRYK WGQGTT VH5 GAEVKKP(SEQ ID GKGLEW SDTRYS TNTAYLQW YFYFGM VTVSS GESLVISC NO: 299) MG (SEQPSFQG SSLKASDTA DV (SEQ (SEQ ID KASGDKD ID (SEQ ID MYYCAR ID NO: 304)TFT (SEQ NO: 300) NO: 301) (SEQ ID NO: 303) ID NO: 298) NO: 302) B6.1QVQLVQS SFWIA WVRQMP IIYAGD HVNISVDRS HDSRYK WGQGTT VH5 GAEVKKP (SEQ IDGKGLEW SDTRYS TNTAYLQW YFYFGM VTVSS GESLVISC NO: 306) MG (SEQ PSFQGSSLKASDTA DV (SEQ (SEQ ID KASGDKD ID (SEQ ID MYYCAR ID NO: 311) TFT (SEQNO: 307) NO: 308) (SEQ ID NO: 310) ID NO: 305) NO: 309) B8 QVQLLES SYGMHWVRQAP VIWYD RFTISRDNSK GYSNYD WGQGTT VH3 GGGVVQP (SEQ ID GKGLEW GSNKYDTLYLQMN YYYGM VTVSS GRSLRLSC NO: 313) VA (SEQ YADSV SLRAEDTAV DV (SEQ(SEQ ID AASGFTFS ID KG YYCAR (SEQ ID NO: 318) (SEQ ID NO: 314) (SEQ IDID NO: 316) NO: 317) NO: 312) NO: 315) B8.1 QVQLLES SYGMH WVRQAP VIWYDRFTISRDNSK GYSNYD WGQGTT VH3 GGGVVQP (SEQ ID GKGLEW GSNKY NTLYLQMN YYYGMVTVSS GRSLRLSC NO: 320) VA (SEQ YADSV SLRAEDTAV DV (SEQ (SEQ ID AASGFTFSID KG YYCAR (SEQ ID NO: 325) (SEQ ID NO: 321) (SEQ ID ID NO: 323)NO: 324) NO: 319) NO: 322) B11 QVQLLQS TYGMH WVRQAP FVSSDG RFTIPRDNADRYPID WGQGTT VH3 AGGVVQP (SEQ ID GKGLEW NNKFY KNTLYLQM CSGGSC VTVSSGRSLRLSC NO: 327) VA (SEQ SDSVK NSLETEDTA FSYGMD (SEQ ID AASGFIFR IDG (SEQ VYYCAK V (SEQ NO: 332) (SEQ ID NO: 328) ID (SEQ ID ID NO: 326)NO: 329) NO: 330) NO: 331) B11C3 EVQLVES TYGMH WVRQAP FVSSDG RFTIPRDNADRYPID WGQGTL VH3 GGGVVQP (SEQ ID GKGLEW NNKFY KNTLYLQM CSGGSC VTVSSGRSLRLSC NO: 334) VA (SEQ SDSVK NSLETEDTA FSYGMD (SEQ ID ATSGFILR IDG (SEQ VYYCAK V NO: 339) (SEQ ID NO: 335) ID (SEQ ID (SEQ ID NO: 333)NO: 336) NO: 337) NO: 338) B11E8 EVQLVQS TYGMH WVRQAP FVSSDG RFTISRDNADRYPID WGQGTT VH3 GGGVVQP (SEQ ID GKGLEW NNKFY KNTLYLQM CSGGSC VTVSSGRSLRLSC NO: 341) VA (SEQ SDSVK NSLETEDTA FSYGMD (SEQ ID AASGFIFR IDG (SEQ MYYCAK V (SEQ NO: 346) (SEQ ID NO: 342) ID (SEQ ID ID NO: 340)NO: 343) NO: 344) NO: 345) B12 QVNLRES SYALH WVRQTP LISYDG RFTISRDNSKDRSHYG WGQGTL VH3 GGGVVQP (SEQ ID GKGLEW SNKYY NMLYLQMN DYVGYL VTVSSGRSLRLSC NO: 348) VA (SEQ ADSVK SLRAEDTAV DY (SEQ (SEQ ID AASGFTFS IDG (SEQ YYCAK (SEQ ID NO: 353) (SEQ ID NO: 349) ID ID NO: 351) NO: 352)NO: 347) NO: 350) B12.1 QVNLRES SYALH WVRQTP LISYDG RFTISRDNSK DRSHYGWGQGTL VH3 GGGVVQP (SEQ ID GKGLEW SNKYY NMLYLQMN DYVGYL VTVSS GRSLRLSCNO: 355) VA (SEQ ADSVK SLRAEDTAV DY (SEQ (SEQ ID AASGFTFS ID G (SEQYYCAK (SEQ ID NO: 360) (SEQ ID NO: 356) ID ID NO: 358) NO: 359) NO: 354)NO: 357) B12.2 QVNLRES SYALH WVRQTP LISYDG RFTISRDNSK DRSHYG WGQGTL VH3GGGVVQP (SEQ ID GKGLEW SNKYY NMLYLQMN DYVGYL VTVSS GRSLRLSC NO: 362)VA (SEQ ADSVK SLRAEDTAV DY (SEQ (SEQ ID AASGFTFS ID G (SEQ YYCAK (SEQ IDNO: 367) (SEQ ID NO: 363) ID ID NO: 365) NO: 366) NO: 361) NO: 364) 1B18EVQLVQS AYWM WVRQAP NINLDG RFTVSRDNV LEWGGR WGQGTL VH3 GGGLVQP T (SEQGKGLEW TEIYYL KNSVFLQMS NGWVSP VTVSS GGSRRLSC ID VA (SEQ DSVKG SLRVEDTAV(SEQ ID (SEQ ID AASGFYF NO: 369) ID (SEQ ID YFCAR (SEQ NO: 373) NO: 374)N (SEQ ID NO: 370) NO: 371) ID NO: 372) NO: 368) 2B18.1 QVQLVQS AYWMWVRQAP NINLDG RFTVSRDNV LEWGGR WGQGTL VH3 GGGLVQP T (SEQ GKGLEW TEIYYLKNSVFLQMS NGWVSP VTVSS GGSRRLSC ID VA (SEQ DSVKG SLRVEDTAV (SEQ ID(SEQ ID AASGFYF NO: 376) ID (SEQ ID YFCAR (SEQ NO: 380) NO: 381)N (SEQ ID NO: 377) NO: 378) ID NO: 379) NO: 375) 4B19 QVQLVQS GYYIYWVRQAP WINPNS RVTMTIDTS EWTQL WGQGTT VH1 GAEVKKP (SEQ ID GQGLEW GVTKYTNTAYMEL WSPYDY VTVSS GASVNVS NO: 383) MG (SEQ AQKFQ NRLRADDT (SEQ ID(SEQ ID CKASGYT ID G (SEQ AVYYCAR NO: 387) NO: 388) FT (SEQ ID NO: 384)ID (SEQ ID NO: 382) NO: 385) NO: 386) 1B22 QVQLQES SYSWS WIRQTPG YIYHSGRVTMSVDK TAFYYE WGQGTL VH4 GSRLVKPS (SEQ ID KGLEWIG STYYN SRNQFSLNMNTGPIRC VTVSS QTLSLTCG NO: 390) (SEQ ID PSLKS SSVTAADTA YLDF (SEQ IDVSGGSISS NO: 391) (SEQ ID VYYCAR (SEQ ID NO: 395) S (SEQ ID NO: 392)(SEQ ID NO: 394) NO: 389) NO: 393)

TABLE 4Deduced protein sequences of light chain variable regions (VL) ofBoNT/B binders. VL/ Clone/ Gene Framework Framework Framework FrameworkFamily 1 CDR1 2 CDR2 3 CDR3 4 A12 DIQMTQSP RASQRI WYQQKP AASSL EVPSRFSGSQQSYRP FGGGTK VK1 SSLSASVG SNYLN GKAPKLL QS (SEQ GSGTDFTLTI PLT (SEQVEIKR DRVTITC (SEQ ID IY (SEQ ID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 397)ID NO: 399) TYYC (SEQ NO: 401) NO: 402) NO: 396) NO: 398) ID NO: 400)6A12 DIQMTQSP RASQGI WYQQKP AASSL GVPSRFSGS QKANSF FGGGTK VK2 SSVSASVGSSWLA GKAPKLL QS (SEQ GSGTDFTLTI PLT (SEQ VEIKR NRVTITC (SEQ ID IY (SEQID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 404) ID NO: 406) TYYC (SEQ NO: 408)NO: 409) NO: 403) NO: 405) ID NO: 407) B1.1 DVVMTQS RASQSI WYQQKP AASSLGVPSRFSGS QQSYST FGQGTKL VK1 PSSLSASV SSYLN GKAPKLL QS (SEQ GSGTDFTLTIPLT (SEQ EIKR GDRVTITC (SEQ ID IY (SEQ ID SSLQPEDFA ID (SEQ ID (SEQ IDNO: 411) ID NO: 413) TYYC (SEQ NO: 415) NO: 416) NO: 410) NO: 412)ID NO: 414) B6 DVVMTQS QAGQD WYQQKP DASNL GVPSRFSGG QQYDNL FGQGTKL VK1PSSLSASV ISNFLN GKAPKLL ET (SEQ GSGTHFTFTI PYT EIKR GDRITITC (SEQ IDIR (SEQ ID SSLHPEDIAT (SEQ ID (SEQ ID (SEQ ID NO: 418) ID NO: 420)TFC (SEQ ID NO: 422) NO: 423) NO: 417) NO: 419) NO: 421) B6.1 DIQMTQSPRASQSI WYQQEP SASSLQ GVPSRFSGS QQSYST FGQGTKP VK1 SSLSASVG SSYLN GKAPKLLS (SEQ GSGTDFTLTI LPYT EIKR DRVTITC (SEQ ID IY (SEQ ID  SSLQPEDFA(SEQ ID (SEQ ID (SEQ ID NO: 425) ID  NO: 427) TYYC (SEQ NO: 429)NO: 430) NO: 424) NO: 426) ID NO: 428) B8 DIQMTQSP RASQRI WYQQKP AASSLEVPSRFSGS QQSYRP FGGGTK VK1 SSLSASVG SNYLN GKAPKLL QS (SEQ GSGTDFTLTIPYT (SEQ VDIKR DRVTITC (SEQ ID IY (SEQ ID  SSLQPEDFA ID (SEQ ID (SEQ IDNO: 432) ID  NO: 434) TYYC (SEQ NO: 436) NO: 437) NO: 431) NO: 433)ID NO: 435) B8.1 DIQMTQSP RASQRI WYQQKP AASSL EVPSRFSGS QQSYRP FGGGTKVK1 SSLSASVG SNYLN GKAPKLL QS (SEQ GYGTDFTLT PLT (SEQ VDIKR DRVTITC(SEQ ID IY (SEQ ID  ISSLQPEDFA ID (SEQ ID (SEQ ID NO: 439) ID  NO: 441)TYYC (SEQ NO: 443) NO: 444) NO: 438) NO: 440) ID NO: 442) B11 DIVMTQSPRASQSI WYQQKP EASSLE GVPSRFSGS QQYDSY FGGGTK VK1 STLSASVG NSWLA GKAPKLLS (SEQ GSGTEFTLTI WLT VEIKR DRVTVTC (SEQ ID IY (SEQ ID  SSLQPDDFA(SEQ ID (SEQ ID (SEQ ID NO: 446) ID  NO: 448) TYYC (SEQ NO: 450)NO: 451) NO: 445) NO: 447) ID NO: 449) B11C3 DIQMTQSP RASQG WYQQRP GASSLGVPSRFSGS QQYDSF FGGGTK VK1 SSVSASVG VSRWL EKAPKLL QS (SEQ GSGTDFTLTIPYT VEIKR DRVTITC A (SEQ IY (SEQ ID  SSLQPEDFA (SEQ ID (SEQ ID (SEQ IDID ID  NO: 455) TYYC (SEQ NO: 457) NO: 458) NO: 452) NO: 453) NO: 454)ID NO: 456) B11E8 EIVLTQSP RASQS WYQQKR GASTR GIPARFSGSG QQYDN FGQGTRLVK1 ATLSVSPG VSKFL GQAPRLL AT (SEQ SGTEFALTIS WPIT EIKR ERATLSC A (SEQIY (SEQ ID  SLQSEDFAD (SEQ ID (SEQ ID (SEQ ID ID ID  NO: 462)YYC (SEQ ID NO: 464) NO: 465) NO: 459) NO: 460) NO: 461) NO: 463) B12DIVMTQSP RASQGI WYQQKP KASSLE GVPSRFSGS LQHNSY FGQGTKL VK1 STLSASVGSSWLA GKAPKLL S (SEQ GSGTEFTLTI PRA EIKR DRVTITC (SEQ ID IY (SEQ ID SSLQPEDFA (SEQ ID (SEQ ID (SEQ ID NO: 467) ID  NO: 469) TYYC (SEQNO: 471) NO: 472) NO: 466) NO: 468) ID NO: 470) B12.1 AYVLTQP EGNNVWYQQRP DDSDR GIPERFSGSN QVWDSS FGGGTKL VL3 PSVSVAPG GNKNV GQAPVL PS (SEQSGNTATLTI SAQWV TVLG KTAAITC H (SEQ VVH (SEQ ID NRVEAGDE (SEQ ID (SEQ ID(SEQ ID ID ID NO: 476) ADYYC (SEQ NO: 478) NO: 479) NO: 473) NO: 474)NO: 475) ID NO: 477) B12.2 ESVLTQPP SGSSSN WYQQLP ENSKRS GIPDRFSGSKGTWDSS FGGGTKL VL1 LVSAAPG IGNNY GTAPKLL S (SEQ SGTSATLGIT LSAVV TVLGQKVTISC VS (SEQ IY (SEQ ID GLQTGDEA (SEQ ID (SEQ ID (SEQ ID ID IDNO: 483) DYYC (SEQ NO: 485) NO: 486) NO: 480) NO: 481) NO: 482)ID NO: 484) 1B18 DVVMTQS RASQSI WYQQRP AASSL AVPSRFSGS QQSYST FGQGTK VK1PSSVSASV SSYLN GKAPKLL QS (SEQ GSGTDFTLTI PPT (SEQ VEIKR GDRVTITC(SEQ ID IF (SEQ ID ID SSLQPEDFA ID (SEQ ID (SEQ ID NO: 488) NO: 489)NO: 490) TYYC (SEQ NO: 492) NO: 493) NO: 487) ID NO: 491) 2B18.1DIVMTQSP RASQSI WYQQKP KTSSLE GVPSRFSGR QQSYST FGGGTK VK1 SSLSASVG SSYLNGKAPKLL S (SEQ GSGTDFTLTI PLT (SEQ VEIKR DRVSISC (SEQ ID IY (SEQ IDSSLQPEDFA ID (SEQ ID (SEQ ID NO: 495) ID NO: 497) TYYC (SEQ NO: 499)NO: 500) NO: 494) NO: 496) ID NO: 498) 1B22 DIQMTQSP RASQSI WYQQRPSASTLQ GVPSRFSGS QQYNSY FGQGTKL VK1 STLSASIG QSWLA GEAPKLL T (SEQGSGTDFTLTI PLT (SEQ EIKR DRVTISC (SEQ ID IY (SEQ ID SSLQPEDFA ID (SEQ ID(SEQ ID NO: 502) ID NO: 504) TYYC (SEQ NO: 506) NO: 507) NO: 501)NO: 503) ID NO: 505) 4B19 DIVLTQSP RASRSI WYQQRP AASSL GVPSRFSGS QQAFGFFGQGTK VK1 STLSASVG GWYLN GKAPKLL HN GSGTEFTLTI PRT (SEQ VEIKR DRVTISC(SEQ ID IY (SEQ (SEQ ID SSLQPDDFA ID (SEQ ID (SEQ ID NO: 509) IDNO: 511) TYYC (SEQ NO: 513) NO: 514) NO: 508) NO: 510) ID NO: 512)

TABLE 5 Unique BoNT/A light chain antibodies. Shown are theclone name, VH CDR3, VL CDR3, KD for BoNT/A lightchain, and epitope recognized. Epitopes areassigned sequential numbers, if the epitopedoes not overlap with other light chain antibodies.Affinities are for BoNT/A1 as determinedusing yeast displayed scFv and soluble BoNT/A1. Clone VH CDR3 VL CDR3 KDEpitope ING2 DPYYYSYMDV QQYYSTPFT 0.25 1 (SEQ ID NO: 515) (SEQ IDNO: 516) 5A20 EASFGWSYLGHDDAFDI QQYGSSLWT 0.34 2 (SEQ IDNO: 517) (SEQ IDNO: 518) CON1 DPGWIYSDTSAAGWFDP QQSYDTPRT 10 3 (4A1.1) (SEQ ID NO: 519)(SEQ ID NO: 520)

Using the teachings and the sequence information provided herein, thevariable light and variable heavy chains can be joined directly orthrough a linker (e.g., (Gly₄Ser)₃, SEQ ID NO:521) to form asingle-chain Fv antibody. The various CDRs and/or framework regions canbe used to form full human antibodies, chimeric antibodies, antibodyfragments, polyvalent antibodies, and the like.

In certain embodiments, the anti-BoNT antibodies of this invention havea binding affinity (K_(D)) for a BoNT protein of at least 10⁻⁸,preferably at least 10⁻⁹, more preferably at least 10⁻¹⁰, and mostpreferably at least 10⁻¹¹, or 10⁻¹².

III. Preparation of BoNT Neutralizing Antibodies.

A) Recombinant Expression of BoNT-Neutralizing Antibodies.

Using the information provided herein, the botulinumneurotoxin-neutralizing antibodies of this invention are prepared usingstandard techniques well known to those of skill in the art.

For example, the polypeptide sequences provided herein (see, e.g.,Tables 1-5, and/or FIGS. 2-3) can be used to determine appropriatenucleic acid sequences encoding the BoNT-neutralizing antibodies and thenucleic acids sequences then used to express one or moreBoNT-neutralizing antibodies. The nucleic acid sequence(s) can beoptimized to reflect particular codon “preferences” for variousexpression systems according to standard methods well known to those ofskill in the art.

Using the sequence information provided, the nucleic acids may besynthesized according to a number of standard methods known to those ofskill in the art. Oligonucleotide synthesis, is preferably carried outon commercially available solid phase oligonucleotide synthesis machines(Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168) ormanually synthesized using, for example, the solid phase phosphoramiditetriester method described by Beaucage et. al. (1981) Tetrahedron Letts.22(20): 1859-1862.

Once a nucleic acid encoding an anti-BoNT antibody is synthesized it canbe amplified and/or cloned according to standard methods. Molecularcloning techniques to achieve these ends are known in the art. A widevariety of cloning and in vitro amplification methods suitable for theconstruction of recombinant nucleic acids are known to persons of skill.Examples of these techniques and instructions sufficient to directpersons of skill through many cloning exercises are found in Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymologyvolume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal. (1989) Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3,Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook);and Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methodsof producing recombinant immunoglobulins are also known in the art. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'lAcad. Sci. USA 86: 10029-10033.

Examples of techniques sufficient to direct persons of skill through invitro amplification methods, including the polymerase chain reaction(PCR) the ligase chain reaction (LCR), Qβ-replicase amplification andother RNA polymerase mediated techniques are found in Berger, Sambrook,and Ausubel, as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202;PCR Protocols A Guide to Methods and Applications (Innis et al. eds)Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson(Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94;(Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173; Guatelli et al.(1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell et al. (1989) J.Clin. Chem 35, 1826; Landegren et al., (1988) Science 241, 1077-1080;Van Brunt (1990) Biotechnology 8, 291-294; Wu and Wallace, (1989) Gene4, 560; and Barringer et al. (1990) Gene 89, 117. Improved methods ofcloning in vitro amplified nucleic acids are described in Wallace etal., U.S. Pat. No. 5,426,039.

Once the nucleic acid for an anti-BoNT antibody is isolated and cloned,one can express the gene in a variety of recombinantly engineered cellsknown to those of skill in the art. Examples of such cells includebacteria, yeast, filamentous fungi, insect (especially employingbaculoviral vectors), and mammalian cells. It is expected that those ofskill in the art are knowledgeable in the numerous expression systemsavailable for expression of antibodies.

In brief summary, the expression of natural or synthetic nucleic acidsencoding anti-BoNT antibodies will typically be achieved by operablylinking a nucleic acid encoding the antibody to a promoter (which iseither constitutive or inducible), and incorporating the construct intoan expression vector. The vectors can be suitable for replication andintegration in prokaryotes, eukaryotes, or both. Typical cloning vectorscontain transcription and translation terminators, initiation sequences,and promoters useful for regulation of the expression of the nucleicacid encoding the anti-BoNT antibody. The vectors optionally comprisegeneric expression cassettes containing at least one independentterminator sequence, sequences permitting replication of the cassette inboth eukaryotes and prokaryotes, i.e., shuttle vectors, and selectionmarkers for both prokaryotic and eukaryotic systems. See Sambrook.

To obtain high levels of expression of a cloned nucleic acid it iscommon to construct expression plasmids which typically contain a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.Examples of regulatory regions suitable for this purpose in E. coli arethe promoter and operator region of the E. coli tryptophan biosyntheticpathway as described by Yanofsky (1984) J. Bacteriol., 158:1018-1024 andthe leftward promoter of phage lambda (PO as described by Herskowitz andHagen (1980) Ann. Rev. Genet., 14:399-445. The inclusion of selectionmarkers in DNA vectors transformed in E. coli is also useful. Examplesof such markers include genes specifying resistance to ampicillin,tetracycline, or chloramphenicol. See Sambrook for details concerningselection markers, e.g., for use in E. coli.

Expression systems for expressing anti-BoNT antibodies are availableusing, for example, E. coli, Bacillus sp. (see, e.g., Palva, et al.(1983) Gene 22:229-235; Mosbach et al. (1983) Nature, 302: 543-545), andSalmonella. In certain embodiments, E. coli systems are preferred.

The anti-BoNT antibodies produced by prokaryotic cells may requireexposure to chaotropic agents for proper folding. During purificationfrom, e.g., E. coli, the expressed protein is optionally denatured andthen renatured. This can be accomplished, e.g., by solubilizing thebacterially produced antibodies in a chaotropic agent such as guanidineHCl. The antibody is then renatured, either by slow dialysis or by gelfiltration (see, e.g., U.S. Pat. No. 4,511,503).

Methods of transfecting and expressing genes in mammalian cells areknown in the art. Transducing cells with nucleic acids can involve, forexample, incubating viral vectors containing anti-BoNT nucleic acidswith cells within the host range of the vector (see, e.g., Goeddel(1990) Methods in Enzymology, vol. 185, Academic Press, Inc., San Diego,Calif. or Krieger (1990) Gene Transfer and Expression—A LaboratoryManual, Stockton Press, New York, N.Y. and the references citedtherein).

The culture of cells used in the present invention, including cell linesand cultured cells from tissue or blood samples is well known in the art(see, e.g., Freshney (1994) Culture of Animal Cells, a Manual of BasicTechnique, third edition, Wiley-Liss, N. Y. and the references citedtherein).

Techniques for using and manipulating antibodies are found in Coligan(1991) Current Protocols in Immunology Wiley/Greene, NY; Harlow and Lane(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY;Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein;Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.)Academic Press, New York, N.Y.; and Kohler and Milstein (1975) Nature256: 495-497.

In one preferred embodiment the BoNT/A-neutralizing antibody gene (e.g.BoNT/A-neutralizing scFv gene) is subcloned into the expression vectorpUC119mycHis (Tomlinson et al. (1996) J. Mol. Biol., 256: 813-817) orpSYN3, resulting in the addition of a hexahistidine tag at theC-terminal end of the scFv to facilitate purification. Detailedprotocols for the cloning and purification of certain BoNT-neutralizingantibodies are found, for example, in Amersdorfer et al. (1997) Infect.Immunity, 65(9): 3743-3752, and the like.

B) Preparation of Whole Polyclonal or Monoclonal Antibodies.

The anti-BoNT antibodies of this invention include individual, allelic,strain, or species variants, and fragments thereof, both in theirnaturally occurring (full-length) forms and in recombinant forms. Incertain embodiments, preferred antibodies are selected to bind one ormore epitopes bound by the antibodies described herein (e.g., 2A10, 3E1,3E2, 3E3, 3E4, 3E4.1, 3E5, 3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17,4E17.1, n A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12,B12.1, B12.2, 1B18, 2B18.1, 4B19, and 1B22). The antibodies can beraised in their native configurations or in non-native configurations.Anti-idiotypic antibodies can also be generated. Many methods of makingantibodies that specifically bind to a particular epitope are known topersons of skill. The following discussion is presented as a generaloverview of the techniques available; however, one of skill willrecognize that many variations upon the following methods are known.

1) Polyclonal Antibody Production.

Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen (e.g., BoNT/A, BoNT/B, BoNT/E, etc.)subsequences including, but not limited to subsequences comprisingepitopes specifically bound by antibodies expressed by clones 2A10, 3E1,3E2, 3E3, 3E4, 3E4.1, 3E5, 3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17,4E17.1, n A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12,B12.1, B12.2, 1B18, 2B18.1, 4B19, and 1B22 disclosed herein, preferablya purified polypeptide, a polypeptide coupled to an appropriate carrier(e.g., GST, keyhole limpet hemanocyanin, etc.), or a polypeptideincorporated into an immunization vector such as a recombinant vacciniavirus (see, U.S. Pat. No. 4,722,848) is mixed with an adjuvant andanimals are immunized with the mixture (see, e.g., FIG. 1). The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the polypeptide ofinterest. When appropriately high titers of antibody to the immunogenare obtained, blood is collected from the animal and antisera areprepared. Further fractionation of the antisera to enrich for antibodiesreactive to the BoNT/A polypeptide is performed where desired (see,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY;and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press, NY).

Antibodies that specifically bind to the neutralizing epitopes describedherein can be selected from polyclonal sera using the selectiontechniques described herein.

2) Monoclonal Antibody Production.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Descriptions of techniques for preparing such monoclonal antibodies arefound in, e.g., Stites et al. (eds.) Basic and Clinical Immunology (4thed.) Lange Medical Publications, Los Altos, Calif., and references citedtherein; Harlow and Lane, supra; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andKohler and Milstein (1975) Nature 256: 495-497.

Summarized briefly, monoclonal antibody production proceeds by injectingan animal with an (e.g., BoNT/A, BoNT/B, BoNT/E, etc.) subsequencesincluding, but not limited to subsequences comprising epitopesspecifically bound by antibodies expressed by clones 2A10, 3E1, 3E2,3E3, 3E4, 3E4.1, 3E5, 3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17,4E17.1, n A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12,B12.1, B12.2, 1B18, 2B18.1, 4B19, and 1B22 disclosed herein. The animalis then sacrificed and cells taken from its spleen, which are fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods knownin the art. Colonies arising from single immortalized cells are screenedfor production of antibodies of the desired specificity and affinity forthe BoNT antigen, and yield of the monoclonal antibodies produced bysuch cells is enhanced by various techniques, including injection intothe peritoneal cavity of a vertebrate (preferably mammalian) host. Theantibodies of the present invention are used with or withoutmodification, and include chimeric antibodies such as humanized murineantibodies.

IV. Modification of BoNT Neutralizing Antibodies.

A) Phage Display can be Used to Increase Antibody Affinity.

To create higher affinity antibodies, mutant scFv gene repertories,based on the sequence of a binding scFv (see, e.g., Tables 1-5, and/orFIGS. 2, and/or 3), can be created and expressed on the surface ofphage. Display of antibody fragments on the surface of viruses whichinfect bacteria (bacteriophage or phage) makes it possible to producehuman or other mammalian antibodies (e.g., scFvs) with a wide range ofaffinities and kinetic characteristics. To display antibody fragments onthe surface of phage (phage display), an antibody fragment gene isinserted into the gene encoding a phage surface protein (e.g., pIII) andthe antibody fragment-pIII fusion protein is expressed on the phagesurface (McCafferty et al. (1990) Nature, 348: 552-554; Hoogenboom etal. (1991) Nucleic Acids Res., 19: 4133-4137).

Since the antibody fragments on the surface of the phage are functional,those phage bearing antigen binding antibody fragments can be separatedfrom non-binding or lower affinity phage by antigen affinitychromatography (McCafferty et al. (1990) Nature, 348: 552-554). Mixturesof phage are allowed to bind to the affinity matrix, non-binding orlower affinity phage are removed by washing, and bound phage are elutedby treatment with acid or alkali. Depending on the affinity of theantibody fragment, enrichment factors of 20 fold-1,000,000 fold areobtained by single round of affinity selection.

By infecting bacteria with the eluted phage or modified variants of theeluted phage as described below, more phage can be grown and subjectedto another round of selection. In this way, an enrichment of 1000 foldin one round becomes 1,000,000 fold in two rounds of selection (see,e.g., McCafferty et al. (1990) Nature, 348: 552-554). Thus, even whenenrichments in each round are low, multiple rounds of affinity selectionleads to the isolation of rare phage and the genetic material containedwithin which encodes the sequence of the binding antibody (see, e.g.,Marks et al. (1991) J. Mol. Biol., 222: 581-597). The physical linkbetween genotype and phenotype provided by phage display makes itpossible to test every member of an antibody fragment library forbinding to antigen, even with libraries as large as 100,000,000 clones.For example, after multiple rounds of selection on antigen, a bindingscFv that occurred with a frequency of only 1/30,000,000 clones wasrecovered (Id).

1) Chain Shuffling.

One approach for creating mutant scFv gene repertoires involvesreplacing either the V_(H) or V_(L) gene from a binding scFv with arepertoire of V_(H) or V_(L) genes (chain shuffling) (see, e.g.,Clackson et al. (1991) Nature, 352: 624-628). Such gene repertoirescontain numerous variable genes derived from the same germline gene asthe binding scFv, but with point mutations (see, e.g., Marks et al.(1992) Bio/Technology, 10: 779-783). Using light or heavy chainshuffling and phage display, the binding avidities of, e.g., BoNT/E orBoNT/B-neutralizing antibody fragment can be dramatically increased(see, e.g., Marks et al. (1992) Bio/Technology, 10: 779-785 in which theaffinity of a human scFv antibody fragment which bound the haptenphenyloxazolone (phox) was increased from 300 nM to 15 nM (20 fold)).

Thus, to alter the affinity of BoNT-neutralizing antibody a mutant scFvgene repertoire is created containing the V_(H) gene of a knownBoNT-neutralizing antibody (e.g., 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1, 3E5,3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, n A12, 6A12, B1.1,B6,136:1, B8, B8.1, BI 1, B11C3, B11E8, B12, B12.1, B12.2, 1B18, 2B18.1,4B19, and 1B22) and a V_(L) gene repertoire (light chain shuffling).Alternatively, an scFv gene repertoire is created containing the V_(L)gene of a known BoNT-neutralizing antibody (e.g 2A10, 3E1, 3E2, 3E3,3E4, 3E4.1, 3E5, 3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, nA12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1,B12.2, 1B18, 2B18.1, 4B19, and 1B22) and a V_(H) gene repertoire (heavychain shuffling). The scFv gene repertoire is cloned into a phagedisplay vector (e.g., pHEN-1, Hoogenboom et al. (1991) Nucleic AcidsRes., 19: 4133-4137) and after transformation a library of transformantsis obtained. Phage are prepared and concentrated and selections areperformed. In addition to chain shuffling, it is also possible toshuffle individual complementarity determining regions (CDRs).

In certain embodiments, the antigen concentration is decreased in eachround of selection, reaching a concentration less than the desired K_(d)by the final rounds of selection. This results in the selection of phageon the basis of affinity (Hawkins et al. (1992) J. Mol. Biol. 226:889-896).

2) Increasing the Affinity of Anti-BoNT Antibodies by Site DirectedMutagenesis.

The majority of antigen contacting amino acid side chains are located inthe complementarity determining regions (CDRs), three in the V_(H)(CDR1, CDR2, and CDR3) and three in the V_(L) (CDR1, CDR2, and CDR3)(see, e.g., Chothia et al. (1987) J. Mol. Biol., 196: 901-917; Chothiaet al. (1986) Science, 233: 755-8; Nhan et al. (1991) J. Mol. Biol.,217: 133-151). Without being bound to a theory, it is believed thatthese residues contribute the majority of binding energetics responsiblefor antibody affinity for antigen. In other molecules, mutating aminoacids that contact ligand has been shown to be an effective means ofincreasing the affinity of one protein molecule for its binding partner(Lowman et al. (1993) J. Mol. Biol., 234: 564-578; Wells (1990)Biochemistry, 29: 8509-8516). Thus mutation (randomization) of the CDRsand screening against, for example, BoNT/A, BoNT/E, BoNT/B, or theepiotpes thereof, can be used to generate anti-BoNT antibodies havingimproved binding affinity.

In certain embodiments, each CDR is randomized in a separate library,using, for example, A 12 as a template. To simplify affinitymeasurement, A12, or other lower affinity anti-BoNT antibodies, are usedas a template, rather than a higher affinity scFv. The CDR sequences ofthe highest affinity mutants from each CDR library are combined toobtain an additive increase in affinity. A similar approach has beenused to increase the affinity of human growth hormone (hGH) for thegrowth hormone receptor over 1500 fold from 3.4×10⁻¹° to 9.0×10⁻¹³ M(see, e.g., Lowman et al. (1993) J. Mol. Biol., 234: 564-578).

To increase the affinity of BoNT-neutralizing antibodies, amino acidresidues located in one or more CDRs (e.g., 9 amino acid residueslocated in V_(L) CDR3) are partially randomized by synthesizing a“doped” oligonucleotide in which the wild type nucleotide occurred witha frequency of, e.g. 49%. The oligonucleotide is used to amplify theremainder of the BoNT-neutralizing scFv gene(s) using PCR.

For example in one embodiment, to create a library in which V_(H) CDR3is randomized an oligonucleotide is synthesized which anneals to theBoNT-neutralizing antibody V_(H) framework 3 and encodes V_(H) CDR3 anda portion of framework 4. At the four positions to be randomized, thesequence NNS can be used, where N is any of the 4 nucleotides, and S is“C” or “T”. The oligonucleotide is used to amplify theBoNT/A-neutralizing antibody V_(H) gene using PCR, creating a mutantBoNT-neutralizing antibody V_(H) gene repertoire. PCR is used to splicethe V_(H) gene repertoire with the BoNT-neutralizing antibody lightchain gene, and the resulting scFv gene repertoire cloned into a phagedisplay vector (e.g., pHEN-1 or pCANTAB5E). Ligated vector DNA is usedto transform electrocompetent E. coli to produce a phage antibodylibrary.

To select higher affinity mutant scFv, each round of selection of thephage antibody libraries is conducted on decreasing amounts of one ormore BoNT subtypes. Clones from the third and fourth round of selectioncan screened for binding to the desired antigen(s) (e.g., BoNT/B BoNT/E,etc.) by ELISA on 96 well plates. scFv from, e.g., twenty to forty ELISApositive clones can be expressed, e.g. in 10 ml cultures, the periplasmharvested, and the scFv k_(off) determined by BIAcore. Clones with theslowest k_(w) are sequenced, and each unique scFv subcloned into anappropriate vector (e.g., pUC119 mycHis). The scFv are expressed inculture, and purified. Affinities of purified scFv can be determined byBIAcore.

By way of illustration, FIG. 7 show a scheme used for affinitymaturation of HuC25 (FIG. 7A) and 3D12 (FIG. 7B) scFv using yeastdisplay (see, e.g.: Ser. No. 11/342,27, filed on Jan. 26, 2006, Ser. No.09/144,886, filed in Aug. 31, 1998, Ser. No. 10/632,706, filed on Aug.1, 2003, and PCT application Nos: PCT/US2006/003070 and PCT/US03/24371,which are incorporated herein by reference for all purposes).

3) Creation of Anti-BoNT (scFv′)2 Homodimers.

To create anti-BoNT (e.g., BoNT-neutralizing) (scFv′)₂ antibodies, twoANTI-BoNT scFvs are joined, either through a linker (e.g., a carbonlinker, a peptide, etc.) or through a disulfide bond between, forexample, two cysteins. Thus, for example, to create disulfide linkedscFv, a cysteine residue can be introduced by site directed mutagenesisbetween a myc tag and a hexahistidine tag at the carboxy-terminus of ananti-BoNT/A. Introduction of the correct sequence can be verified by DNAsequencing. In certain embodiments, the construct is in pUC119, so thatthe pelB leader directs expressed scFv to the periplasm and cloningsites (Ncol and Notl) exist to introduce anti-BoNT mutant scFv.Expressed scFv has the myc tag at the C-terminus, followed by twoglycines, a cysteine, and then 6 histidines to facilitate purificationby IMAC. After disulfide bond formation between the two cysteineresidues, the two scFv can be separated from each other by 26 aminoacids (two 11 amino acid myc tags and 4 glycines). An scFv was expressedfrom this construct, purified by IMAC may predominantly comprisemonomeric scFv. To produce (scFv′)₂ dimers, the cysteine can be reducedby incubation with 1 MM beta-mercaptoethanol, and half of the scFvblocked by the addition of DTNB. Blocked and unblocked scFvs can beincubated together to form (scFv′)₂ and the resulting material canoptionally be analyzed by gel filtration. The affinity of the anti-BoNTscFv′ monomer and (scFv′)₂ dimer can optionally be determined byBIAcore.

In certain embodiments, the (scFv′)₂ dimer is created by joining thescFv fragments through a linker, more preferably through a peptidelinker. This can be accomplished by a wide variety of means well knownto those of skill in the art. For example, one preferred approach isdescribed by Holliger et al. (1993) Proc. Natl. Acad. Sci. USA, 90:6444-6448 (see also WO 94/13804).

Typically, linkers are introduced by PCR cloning. For example, syntheticoligonucleotides encoding the 5 amino acid linker (Gly₄Ser, SEQ IDNO:522) can be used to PCR amplify the BoNT/A-neutralizing antibodyV_(H) and V_(L) genes which are then spliced together to create theBoNT/A-neutralizing diabody gene. The gene can then be cloned into anappropriate vector, expressed, and purified according to standardmethods well known to those of skill in the art.

4) Preparation of anti-BoNT (scFv)₂, Fab, and Fab′ Molecules.

Anti-BoNT antibodies such as anti-BoNT/E or anti-BoN/B scFv, orvariant(s) with higher affinity, are suitable templates for creatingsize and valency variants. For example, an anti-BoNT (scFv′)₂ can becreated from the parent scFv (e.g., 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1,3E5, 3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, n A12, 6A12,B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2, 1B18,2B18.1, 4B19, 1B22, etc.) as described above. An scFv gene can beexcised using appropriate restriction enzymes and cloned into anothervector as described herein.

In one embodiment, expressed scFv has a myc tag at the C-terminus,followed by two glycines, a cysteine, and six histidines to facilitatepurification. In certain embodiments, after disulfide bond formationbetween the two cystine residues, the two scFv are separated from eachother by 26 amino acids (e.g., two eleven amino acid myc tags and fourglycines). Single-chain Fv (scFv) can be expressed from this constructand purified.

To produce (scFv′)₂ dimers, the cysteine is reduced by incubation with 1mM β-mercaptoethanol, and half of the scFv blocked by the addition ofDTNB. Blocked and unblocked scFv are incubated together to form(scFv′)₂, which is purified. As higher affinity scFv are isolated, theirgenes are similarly used to construct (scFv′)₂.

In certain embodiments, anti-BoNT Fab are expressed in E. coli using anexpression vector similar to the one described by Better et. al. (1988)Science, 240: 1041-1043. For example, to create a BoNT/B orBoNT/E-neutralizing Fab, the V_(H) and V_(L) genes are amplified fromthe scFv using PCR. The V_(H) gene is cloned into an expression vector(e.g., a PUC119 based bacterial expression vector) that provides an IgGC_(H)1 domain downstream from, and in frame with, the V_(H) gene. Thevector also contains the lac promoter, a pelb leader sequence to directexpressed V_(H)-C_(H)1 domain into the periplasm, a gene 3 leadersequence to direct expressed light chain into the periplasm, and cloningsites for the light chain gene. Clones containing the correct VH geneare identified, e.g., by PCR fingerprinting. The V_(L) gene is splicedto the C_(L) gene using PCR and cloned into the vector containing theV_(H) C_(H)1 gene.

B) Selection of Neutralizing Antibodies.

In certain embodiments, selection of anti-BoNT antibodies (whetherproduced by phage display, yeast display, immunization methods,hybridoma technology, etc.) involves screening the resulting antibodiesfor specific binding to an appropriate antigen(s). In the instant case,suitable antigens can include, but are not limited to BoNT/E, BoNT/B,BoNT/A1, BoNT/A2, BoNT/A3 H_(C), a C-terminal domain of BoNT heavy chain(binding domain), BoNT/A3 holotoxins, r recombinant BoNT domains such asHC (binding domain), HN (translocation domain), or LC (light chain), andthe like. In certain embodiments the neutralizing antibodies areselected for specific binding of an epitope recognized by one or more ofthe antibodies described herein.

Selection can be by any of a number of methods well known to those ofskill in the art. In an illustrative embodiment, selection is byimmunochromatography (e.g., using immunotubes, Maxisorp, Nunc) againstthe desired target, e.g., BoNT/E, BoNT/B, etc. In another embodiment,selection is against a BoNT protein in a surface plasmon resonancesystem (e.g., BIAcore, Pharmacia) either alone or in combination with anantibody that binds to an epitope specifically bound by one or more ofthe antibodies described herein. Selection can also be done using flowcytometry for yeast display libraries. In one embodiment, yeast displaylibraries are sequentially selected, first on BoNT/A1, then on BoNT/A2to obtain antibodies that bind with high affinity to both subtypes ofBoNT/A. This can be repeated for other subtypes.

For phage display, analysis of binding can be simplified by including anamber codon between the antibody fragment gene and gene III. This makesit possible to easily switch between displayed and soluble antibodyfragments simply by changing the host bacterial strain. When phage aregrown in a supE suppresser strain of E. coli, the amber stop codonbetween the antibody gene and gene III is read as glutamine and theantibody fragment is displayed on the surface of the phage. When elutedphage are used to infect a non-suppressor strain, the amber codon isread as a stop codon and soluble antibody is secreted from the bacteriainto the periplasm and culture media (Hoogenboom et al. (1991) NucleicAcids Res., 19: 4133-4137). Binding of soluble scFv to antigen can bedetected, e.g., by ELISA using a murine IgG monoclonal antibody (e.g.,9E10) which recognizes a C-terminal myc peptide tag on the scFv (Evan etal. (1985) Mol. Cell Biol., 5: 3610-3616; Munro et al. (1986) Cell, 46:291-300), e.g., followed by incubation with polyclonal anti-mouse Fcconjugated to a detectable label (e.g., horseradish peroxidase).

As indicated above, purification of the anti-BoNT antibody can befacilitated by cloning of the scFv gene into an expression vector (e.g.,expression vector pUC119mycHIS) that results in the addition of the mycpeptide tag followed by a hexa-histidine tag at the C-terminal end ofthe scFv. The vector also preferably encodes the pectate lyase leadersequence that directs expression of the scFv into the bacterialperiplasm where the leader sequence is cleaved. This makes it possibleto harvest native properly folded scFv directly from the bacterialperiplasm. The BoNT-neutralizing antibody is then expressed and purifiedfrom the bacterial supernatant using immobilized metal affinitychromatography.

C) Measurement of Anti-BoNT Antibody Affinity for One or More BoNTSubtypes.

As explained above, selection for increased avidity involves measuringthe affinity of an anti-BoNT (e.g., a BoNT-neutralizing) antibody (or amodified BoNT-neutralizing antibody) for one or more targets of interest(e.g. BoNT/E subtype(s) or domains thereof. For example, the K_(d) of aBoNT/E-neutralizing antibody and the kinetics of binding to BoNT/E aredetermined in a BIAcore, a biosensor based on surface plasmon resonance.For this technique, antigen is coupled to a derivatized sensor chipcapable of detecting changes in mass. When antibody is passed over thesensor chip, antibody binds to the antigen resulting in an increase inmass that is quantifiable. Measurement of the rate of association as afunction of antibody concentration can be used to calculate theassociation rate constant (k_(on)). After the association phase, bufferis passed over the chip and the rate of dissociation of antibody(k_(off)) determined. K_(on), is typically measured in the range 1.0×10²to 5.0×10⁶ and k_(off) in the range 1.0×10⁻¹ to 1.0×10⁻⁶. Theequilibrium constant K_(d) is then calculated as k_(off)/k_(on) and thusis typically measured in the range 10⁻⁵ to 10⁻¹². Affinities measured inthis manner correlate well with affinities measured in solution byfluorescence quench titration.

Phage display and selection generally results in the selection of higheraffinity mutant scFvs (Marks et al. (1992) Bio/Technology, 10: 779-783;Hawkins et al. (1992) 1 Mol. Biol. 226: 889-896; Riechmann et al. (1993)Biochemistry, 32: 8848-8855; Clackson et al. (1991) Nature, 352:624-628), but probably does not result in the separation of mutants withless than a 6 fold difference in affinity (Riechmann et al. (1993)Biochemistry, 32: 8848-8855). Thus a rapid method is needed to estimatethe relative affinities of mutant scFvs isolated after selection. Sinceincreased affinity results primarily from a reduction in the k_(off),measurement of k_(off) should identify higher affinity scFv. k_(off) canbe measured in the BIAcore on unpurified scFv in bacterial periplasm,since expression levels are high enough to give an adequate bindingsignal and k_(off) is independent of concentration. The value of k_(off)for periplasmic and purified scFv is typically in close agreement.

V. Human or Humanized (Chimeric) Antibody Production.

As indicated above, the anti-BoNT antibodies of this invention can beadministered to an organism (e.g., a human patient) for therapeuticpurposes (e.g., the treatment of botulism). Antibodies administered toan organism other than the species in which they are raised can beimmunogenic. Thus, for example, murine antibodies repeatedlyadministered to a human often induce an immunologic response against theantibody (e.g., the human anti-mouse antibody (HAMA) response). Whilethis is typically not a problem for the use of non-human antibodies ofthis invention as they are typically not utilized repeatedly, theimmunogenic properties of the antibody are reduced by altering portions,or all, of the antibody into characteristically human sequences therebyproducing chimeric or human antibodies, respectively.

A) Chimeric Antibodies.

Chimeric) antibodies are immunoglobulin molecules comprising a human andnon-human portion. More specifically, the antigen combining region (orvariable region) of a chimeric antibody is derived from a non-humansource (e.g., murine) and the constant region of the chimeric antibody(which confers biological effector function to the immunoglobulin) isderived from a human source. The chimeric antibody should have theantigen binding specificity of the non-human antibody molecule and theeffector function conferred by the human antibody molecule. A largenumber of methods of generating chimeric antibodies are well known tothose of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167,5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867,5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and4,975,369).

In general, the procedures used to produce chimeric antibodies consistof the following steps (the order of some steps may be interchanged):(a) identifying and cloning the correct gene segment encoding theantigen binding portion of the antibody molecule; this gene segment(known as the VDJ, variable, diversity and joining regions for heavychains or VJ, variable, joining regions for light chains (or simply asthe V or variable region) may be in either the cDNA or genomic form; (b)cloning the gene segments encoding the constant region or desired partthereof; (c) ligating the variable region to the constant region so thatthe complete chimeric antibody is encoded in a transcribable andtranslatable form; (d) ligating this construct into a vector containinga selectable marker and gene control regions such as promoters,enhancers and poly(A) addition signals; (e) amplifying this construct ina host cell (e.g., bacteria); (f) introducing the DNA into eukaryoticcells (transfection) most often mammalian lymphocytes; and culturing thehost cell under conditions suitable for expression of the chimericantibody.

Antibodies of several distinct antigen binding specificities have beenmanipulated by these protocols to produce chimeric proteins (e.g.,anti-TNP: Boulianne et al. (1984) Nature, 312: 643; and anti-tumorantigens: Sahagan et al. (1986) J. Immunol., 137: 1066). Likewiseseveral different effector functions have been achieved by linking newsequences to those encoding the antigen binding region. Some of theseinclude enzymes (Neuberger et al. (1984) Nature 312: 604),immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain (Sharon et al. (1984) Nature309: 364; Tan et al., (1985) J. Immunol. 135: 3565-3567).

In one preferred embodiment, a recombinant DNA vector is used totransfect a cell line that produces an anti-BoNT antibody. The novelrecombinant DNA vector contains a “replacement gene” to replace all or aportion of the gene encoding the immunoglobulin constant region in thecell line (e.g., a replacement gene may encode all or a portion of aconstant region of a human immunoglobulin, a specific immunoglobulinclass, or an enzyme, a toxin, a biologically active peptide, a growthfactor, inhibitor, or a linker peptide to facilitate conjugation to adrug, toxin, or other molecule, etc.), and a “target sequence” whichallows for targeted homologous recombination with immunoglobulinsequences within the antibody producing cell.

In another embodiment, a recombinant DNA vector is used to transfect acell line that produces an antibody having a desired effector function,(e.g., a constant region of a human immunoglobulin) in which case, thereplacement gene contained in the recombinant vector may encode all or aportion of a region of an BoNT/A-neutralizing antibody and the targetsequence contained in the recombinant vector allows for homologousrecombination and targeted gene modification within the antibodyproducing cell. In either embodiment, when only a portion of thevariable or constant region is replaced, the resulting chimeric antibodymay define the same antigen and/or have the same effector function yetbe altered or improved so that the chimeric antibody may demonstrate agreater antigen specificity, greater affinity binding constant,increased effector function, or increased secretion and production bythe transfected antibody producing cell line, etc.

Regardless of the embodiment practiced, the processes of selection forintegrated DNA (via a selectable marker), screening for chimericantibody production, and cell cloning, can be used to obtain a clone ofcells producing the chimeric antibody.

Thus, a piece of DNA which encodes a modification for a monoclonalantibody can be targeted directly to the site of the expressedimmunoglobulin gene within a B-cell or hybridoma cell line. DNAconstructs for any particular modification may be used to alter theprotein product of any monoclonal cell line or hybridoma. Such aprocedure circumvents the costly and time consuming task of cloning bothheavy and light chain variable region genes from each B-cell cloneexpressing a useful antigen specificity. In addition to circumventingthe process of cloning variable region genes, the level of expression ofchimeric antibody should be higher when the gene is at its naturalchromosomal location rather than at a random position. Detailed methodsfor preparation of chimeric (humanized) antibodies can be found in U.S.Pat. No. 5,482,856.

B) Human and Humanized Antibodies.

In another embodiment, this invention provides for humanized or fullyhuman anti-BoNT-neutralizing antibodies (e.g., 2A10, 3E1, 3E2, 3E3, 3E4,3E4.1, 3E5, 3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, n A12,6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2,1B18, 2B18.1, 4B19, and 1B22, etc.). Human antibodies consist entirelyof characteristically human polypeptide sequences. The humanBoNT-neutralizing antibodies of this invention can be produced in usinga wide variety of methods (see, e.g., Larrick et al., U.S. Pat. No.5,001,065, for review).

In certain preferred embodiments, fully human scFv antibodies of thisinvention are obtained by modification and screening of fully humansingle-chain (e.g. scFv) libraries. Thus, in certain embodiments, fullyhuman antibodies are produced using phage and/or yeast display methodsas described herein. Methods of producing fully human gene libraries arewell known to those of skill in the art (see, e.g., Vaughn et al. (1996)Nature Biotechnology, 14(3): 309-314, Marks et al. (1991) J. Mol. Biol.,222: 581-597, and PCT/US96/10287).

In another embodiment, human BoNT-neutralizing antibodies of the presentinvention are can be produced in trioma cells. Genes encoding theantibodies are then cloned and expressed in other cells, particularly,nonhuman mammalian cells.

The general approach for producing human antibodies by trioma technologyhas been described by Ostberg et al. (1983) Hybridoma 2: 361-367,Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No.4,634,666. The antibody-producing cell lines obtained by this method arecalled triomas because they are descended from three cells; two humanand one mouse. Triomas have been found to produce antibody more stablythan ordinary hybridomas made from human cells.

Preparation of trioma cells requires an initial fusion of a mousemyeloma cell line with unimmunized human peripheral B lymphocytes. Thisfusion generates a xenogenic hybrid cell containing both human and mousechromosomes (see, Engelman, supra.). Xenogenic cells that have lost thecapacity to secrete antibodies are selected. Preferably, a xenogeniccell is selected that is resistant to 8-azaguanine. Such cells areunable to propagate on hypoxanthine-aminopterin-thymidine (HAT) orazaserine-hypoxanthine (AH) media.

The capacity to secrete antibodies is conferred by a further fusionbetween the xenogenic cell and B-lymphocytes immunized against a BoNTpolypeptide (e.g., BoNT/A, BoNT/A H_(c), BoNT/A subsequences including,but not limited to subsequences comprising epitopes specifically boundby the antibodies described herein, etc.). The B-lymphocytes areobtained from the spleen, blood or lymph nodes of human donor. Ifantibodies against a specific antigen or epitope are desired, it ispreferable to use that antigen or epitope thereof as the immunogenrather than the entire polypeptide. Alternatively, B-lymphocytes areobtained from an unimmunized individual and stimulated with a BoNTpolypeptide, or a epitope thereof, in vitro. In a further variation,B-lymphocytes are obtained from an infected, or otherwise immunizedindividual, and then hyperimmunized by exposure to a BoNT polypeptidefor about seven to fourteen days, in vitro.

The immunized B-lymphocytes prepared by one of the above procedures arefused with a xenogenic hybrid cell by well known methods. For example,the cells are treated with 40-50% polyethylene glycol of MW 1000-4000,at about 37° C. for about 5-10 min. Cells are separated from the fusionmixture and propagated in media selective for the desired hybrids. Whenthe xenogenic hybrid cell is resistant to 8-azaguanine, immortalizedtrioma cells are conveniently selected by successive passage of cells onHAT or AH medium. Other selective procedures are, of course, possibledepending on the nature of the cells used in fusion. Clones secretingantibodies having the required binding specificity are identified byassaying the trioma culture medium for the ability to bind to the BoNTpolypeptide or an epitope thereof. Triomas producing human antibodieshaving the desired specificity are subcloned by the limiting dilutiontechnique and grown in vitro in culture medium, or are injected intoselected host animals and grown in vivo.

The trioma cell lines obtained are then tested for the ability to bind aBoNT polypeptide or an epitope thereof. Antibodies are separated fromthe resulting culture medium or body fluids by conventionalantibody-fractionation procedures, such as ammonium sulfateprecipitation, DEAE cellulose chromatography and affinitychromatography.

Although triomas are genetically stable they do not produce antibodiesat very high levels. Expression levels can be increased by cloningantibody genes from the trioma into one or more expression vectors, andtransforming the vector into a cell line such as the cell linestypically used for expression of recombinant or humanizedimmunoglobulins. As well as increasing yield of antibody, this strategyoffers the additional advantage that immunoglobulins are obtained from acell line that does not have a human component, and does not thereforeneed to be subjected to the especially extensive viral screeningrequired for human cell lines.

The genes encoding the heavy and light chains of immunoglobulinssecreted by trioma cell lines are cloned according to methods, includingbut not limited to, the polymerase chain reaction (PCR), known in theart (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd ed., Cold Spring Harbor, N.Y., 1989; Berger & Kimmel, Methods inEnzymology, Vol. 152: Guide to Molecular Cloning Techniques, AcademicPress, Inc., San Diego, Calif., 1987; Co et al. (1992) J. Immunol., 148:1149). For example, genes encoding heavy and light chains are clonedfrom a trioma's genomic DNA or cDNA produced by reverse transcription ofthe trioma's RNA. Cloning is accomplished by conventional techniquesincluding the use of PCR primers that hybridize to the sequencesflanking or overlapping the genes, or segments of genes, to be cloned.

Typically, recombinant constructs comprise DNA segments encoding acomplete human immunoglobulin heavy chain and/or a complete humanimmunoglobulin light chain of an immunoglobulin expressed by a triomacell line. Alternatively, DNA segments encoding only a portion of theprimary antibody genes are produced, which portions possess bindingand/or effector activities. Other recombinant constructs containsegments of trioma cell line immunoglobulin genes fused to segments ofother immunoglobulin genes, particularly segments of other humanconstant region sequences (heavy and/or light chain). Human constantregion sequences can be selected from various reference sources,including but not limited to those listed in Kabat et al. (1987)Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services.

In addition to the DNA segments encoding anti-BoNT immunoglobulins orfragments thereof, other substantially homologous modifiedimmunoglobulins can be readily designed and manufactured utilizingvarious recombinant DNA techniques known to those skilled in the artsuch as site-directed mutagenesis (see Gillman & Smith (1979) Gene, 8:81-97; Roberts et al. (1987) Nature 328: 731-734). Such modifiedsegments will usually retain antigen binding capacity and/or effectorfunction. Moreover, the modified segments are usually not so far changedfrom the original trioma genomic sequences to prevent hybridization tothese sequences under stringent conditions. Because, like many genes,immunoglobulin genes contain separate functional regions, each havingone or more distinct biological activities, the genes may be fused tofunctional regions from other genes to produce fusion proteins (e.g.,immunotoxins) having novel properties or novel combinations ofproperties.

The genomic sequences can be cloned and expressed according to standardmethods as described herein.

Other approaches to antibody production include in vitro immunization ofhuman blood. In this approach, human blood lymphocytes capable ofproducing human antibodies are produced. Human peripheral blood iscollected from the patient and is treated to recover mononuclear cells.The suppressor T-cells then are removed and remaining cells aresuspended in a tissue culture medium to which is added the antigen andautologous serum and, preferably, a nonspecific lymphocyte activator.The cells then are incubated for a period of time so that they producethe specific antibody desired. The cells then can be fused to humanmyeloma cells to immortalize the cell line, thereby to permit continuousproduction of antibody (see U.S. Pat. No. 4,716,111).

In another approach, mouse-human hybridomas which produce humanBoNT-neutralizing antibodies are prepared (see, e.g., U.S. Pat. No.5,506,132). Other approaches include immunization of murines transformedto express human immunoglobulin genes, and phage display screening(Vaughan et al. supra.).

Vi. Assaying for Cross-Reactivity at a Neutralizing Epitope.

In a preferred embodiment, the antibodies of this invention specificallybind to one or more epitopes recognized by antibodies described herein(e.g., 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1, 3E5, 3E6, 3E6.1, 4E11, 4E13,4E16, 4E16.1, 4E17, 4E17.1, n A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11,B11C3, B11E8, B12, B12.1, B12.2, 1B18, 2B18.1, 4B19, and 1B22, etc.). Inother words, particularly preferred antibodies are cross-reactive withone of more of these antibodies. Means of assaying for cross-reactivityare well known to those of skill in the art (see, e.g., Dowbenko et al.(1988), J. Virol. 62: 4703-4711).

This can be ascertained by providing one or more isolated target BoNTpolypeptide(s) (e.g. BoNT/A1 and/or BoNT/A2, or recombinant domains ofsaid toxin, such as H_(c)) attached to a solid support and assaying theability of a test antibody to compete with, e.g., 2A10, 3E1, 3E2, 3E3,3E4, 3E4.1, 3E5, 3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, nA12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1,B12.2, 1B18, 2B18.1, 4B19, and 1B22, etc for binding to the target BoNTpeptide. Thus, immunoassays in a competitive binding format arepreferably used for crossreactivity determinations. For example, in oneembodiment, a BoNT/E and/or BoNT/B polypeptide is immobilized to a solidsupport. Antibodies to be tested (e.g. generated by selection from aphage-display library) added to the assay compete with 2A10, 3E1, 3E2,3E3, 3E4, 3E4.1, 3E5, 3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17,4E17.1, n A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12,B12.1, B12.2, 1B18, 2B18.1, 4B19, and 1B22, etc antibodies binding tothe immobilized BoNT polypeptide(s). The ability of test antibodies tocompete with the binding of the 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1, 3E5,3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, n A12, 6A12, B1.1,B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2, 1B18, 2B18.1,4B19, and 1B22, etc antibodies to the immobilized protein(s) arecompared. The percent crossreactivity above proteins is then calculated,using standard calculations.

If the test antibody competes with one or more of the 2A10, 3E1, 3E2,3E3, 3E4, 3E4.1, 3E5, 3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17,4E17.1, n A12, 6A12, B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12,B12.1, B12.2, 1B18, 2B18.1, 4B19, and 1B22, etc antibodies and has abinding affinity comparable to or greater than about 1×10⁻⁸ M with thesame target then the test antibody is expected to be a BoNT-neutralizingantibody.

In a particularly preferred embodiment, cross-reactivity is performed byusing surface plasmon resonance in a BIAcore. In a BIAcore flow cell,the BoNT polypeptide(s) (e.g., BoNT/B and/or BoNT/E) are coupled to asensor chip (e.g. CM5) as described in the examples. With a flow rate of5 μl/min, a titration of 100 nM to 1 μM antibody is injected over theflow cell surface for about 5 minutes to determine an antibodyconcentration that results in near saturation of the surface. Epitopemapping or cross-reactivity is then evaluated using pairs of antibodiesat concentrations resulting in near saturation and at least 100 RU ofantibody bound. The amount of antibody bound is determined for eachmember of a pair, and then the two antibodies are mixed together to givea final concentration equal to the concentration used for measurementsof the individual antibodies. Antibodies recognizing different epitopesshow an essentially additive increase in the RU bound when injectedtogether, while antibodies recognizing identical epitopes show only aminimal increase in RU (see the examples). In a particularly preferredembodiment, antibodies are said to be cross-reactive if, when “injected”together they show an essentially additive increase (preferably anincrease by at least a factor of about 1.4, more preferably an increaseby at least a factor of about 1.6, and most preferably an increase by atleast a factor of about 1.8 or 2.

Cross-reactivity at the desired epitopes can ascertained by a number ofother standard techniques (see, e.g., Geysen et al (1987) J. Immunol.Meth. 102, 259-274). This technique involves the synthesis of largenumbers of overlapping BoNT peptides. The synthesized peptides are thenscreened against one or more of the prototypical antibodies (e.g., CR1,RAZ1, ING1, ING2, etc.) and the characteristic epitopes specificallybound by these antibodies can be identified by binding specificity andaffinity. The epitopes thus identified can be conveniently used forcompetitive assays as described herein to identify cross-reactingantibodies.

The peptides for epitope mapping can be conveniently prepared using“Multipin” peptide synthesis techniques (see, e.g., Geysen et al (1987)Science, 235: 1184-1190). Using the known sequence of one or more BoNTsubtypes (see, e.g., Atassi et al. (1996) J. Prot. Chem., 7: 691-700 andreferences cited therein), overlapping BoNT polypeptide sequences can besynthesized individually in a sequential manner on plastic pins in anarray of one or more 96-well microtest plate(s).

The procedure for epitope mapping using this multipin peptide system isdescribed in U.S. Pat. No. 5,739,306. Briefly, the pins are firsttreated with a pre-coat buffer containing 2% bovine serum albumin and0.1% Tween 20 in PBS for 1 hour at room temperature. Then the pins arethen inserted into the individual wells of 96-well microtest platecontaining the antibodies in the pre-coat buffer, e.g. at 2 μg/ml. Theincubation is preferably for about 1 hour at room temperature. The pinsare washed in PBST (e.g., 3 rinses for every 10 minutes), and thenincubated in the wells of a 96-well microtest plate containing 100 mu 1of HRP-conjugated goat anti-mouse IgG (Fc) (Jackson ImmunoResearchLaboratories) at a 1:4,000 dilution for 1 hour at room temperature.After the pins are washed as before, the pins are put into wellscontaining peroxidase substrate solution of diammonium2,2′-azino-bis[3-ethylbenzthiazoline-b-sulfonate] (ABTS) and H₂O₂(Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) for 30 minutesat room temperature for color reaction. The plate is read at 405 nm by aplate reader (e.g., BioTek ELISA plate reader) against a backgroundabsorption wavelength of 492 nm. Wells showing color developmentindicated reactivity of the BoNT/A H_(C) peptides in such wells withS25, C25, C39, 1C6, or 1F3 antibodies.

VII. Assaying for Neutralizing Activity of Anti-BoNT Antibodies.

Preferred antibodies of this invention act, individually or incombination, to neutralize (reduce or eliminate) the toxicity ofbotulinum neurotoxin type. Neutralization can be evaluated in vivo or invitro. In vivo neutralization measurements simply involve measuringchanges in the lethality (e.g., LD₅₀ or other standard metric) due to aBoNT neurotoxin administration due to the presence of one or moreantibodies being tested for neutralizing activity. The neurotoxin can bedirectly administered to the test organism (e.g. mouse) or the organismcan harbor a botulism infection (e.g., be infected with Clostridiumbotulinum). The antibody can be administered before, during, or afterthe injection of BoNT neurotoxin or infection of the test animal. Adecrease in the rate of progression, or mortality rate indicates thatthe antibody(s) have neutralizing activity.

One suitable in vitro assay for neutralizing activity uses ahemidiaphragm preparation (Deshpande et al. (1995) Toxicon, 33:551-557). Briefly, left and right phrenic nerve hemidiaphragmpreparations are suspended in physiological solution and maintained at aconstant temperature (e.g. 36° C.). The phrenic nerves are stimulatedsupramaximally (e.g. at 0.05 Hz with square waves of 0.2 ms duration).Isometric twitch tension is measured with a force displacementtransducer (e.g., GrassModel FT03) connected to a chart recorder.

Purified antibodies are incubated with purified BoNT (e.g. BoNT/A1,BoNT/A2, BoNT/B, etc.) for 30 min at room temperature and then added tothe tissue bath, resulting in a final antibody concentration of about2.0×10⁻⁸ M and a final BoNT concentration of about 2.0×10⁻¹¹ M. For eachantibody studied, time to 50% twitch tension reduction is determined(e.g., three times for BoNT alone and three times for antibody plusBoNT). Differences between times to a given (arbitrary) percentage (e.g.50%) twitch reduction are determined by standard statistical analyses(e.g. two-tailed t test) at standard levels of significance (e.g., a Pvalue of <0.05 considered significant).

VIII. Diagnostic Assays.

As explained above, the anti-BoNT antibodies of this invention can beused for the in vivo or in vitro detection of BoNT toxin (e.g. BoNT/Etoxin) and thus, are useful in the diagnosis (e.g. confirmatorydiagnosis) of botulism. The detection and/or quantification of BoNT in abiological sample obtained from an organism is indicative of aClostridium botulinum infection of that organism.

The BoNT antigen can be quantified in a biological sample derived from apatient such as a cell, or a tissue sample derived from a patient. Asused herein, a biological sample is a sample of biological tissue orfluid that contains a BoNT concentration that may be correlated with andindicative of a Clostridium botulinum infection. Preferred biologicalsamples include blood, urine, saliva, and tissue biopsies.

Although the sample is typically taken from a human patient, the assayscan be used to detect BoNT antigen in cells from mammals in general,such as dogs, cats, sheep, cattle and pigs, and most particularlyprimates such as humans, chimpanzees, gorillas, macaques, and baboons,and rodents such as mice, rats, and guinea pigs.

Tissue or fluid samples are isolated from a patient according tostandard methods well known to those of skill in the art, most typicallyby biopsy or venipuncture. The sample is optionally pretreated asnecessary by dilution in an appropriate buffer solution or concentrated,if desired. Any of a number of standard aqueous buffer solutions,employing one of a variety of buffers, such as phosphate, Tris, or thelike, at physiological pH can be used.

A) Immunological Binding Assays

The BoNT polypeptide (e.g., BoNT/E, BoNT/B, etc.) can be detected in animmunoassay utilizing one or more of the anti-BoNT antibodies of thisinvention as a capture agent that specifically binds to the BoNTpolypeptide.

As used herein, an immunoassay is an assay that utilizes an antibody(e.g. a anti-BoNT/E antibody) to specifically bind an analyte (e.g.,BoNT/E). The immunoassay is characterized by the binding of one or moreanti-BoNT antibodies to a target (e.g. one or more BoNT/A subtypes) asopposed to other physical or chemical properties to isolate, target, andquantify the BoNT analyte.

The BoNT marker can be detected and quantified using any of a number ofwell recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168, and the like) For areview of the general immunoassays, see also Methods in Cell BiologyVolume 37: Antibodies in Cell Biology, Asai, ed. Academic Press, Inc.New York (1993); Basic and Clinical Immunology 7th Edition, Stites &Ten, eds. (1991)).

The immunoassays of the present invention can be performed in any of anumber of configurations (see, e.g., those reviewed in Maggio (ed.)(1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.; Tijan (1985)“Practice and Theory of Enzyme Immunoassays,” Laboratory Techniques inBiochemistry and Molecular Biology, Elsevier Science Publishers B.V.,Amsterdam; Harlow and Lane, supra; Chan (ed.) (1987) Immunoassay: APractical Guide Academic Press, Orlando, Fla.; Price and Newman (eds.)(1991) Principles and Practice of Immunoassays Stockton Press, NY; andNgo (ed.) (1988) Non isotopic Immunoassays Plenum Press, NY).

Immunoassays often utilize a labeling agent to specifically bind to andlabel the binding complex formed by the capture agent and the analyte(e.g., an anti-BoNT/E antibody/BoNT/E complex). The labeling agent canitself be one of the moieties comprising the antibody/analyte complex.Thus, for example, the labeling agent can be a labeled BoNT/Epolypeptide or a labeled anti-BoNT/E antibody. Alternatively, thelabeling agent is optionally a third moiety, such as another antibody,that specifically binds to the BoNT antibody, the BoNT peptide(s), theantibody/polypeptide complex, or to a modified capture group (e.g.,biotin) which is covalently linked to BoNT polypeptide or to theanti-BoNT antibody.

In one embodiment, the labeling agent is an antibody that specificallybinds to the anti-BoNT antibody. Such agents are well known to those ofskill in the art, and most typically comprise labeled antibodies thatspecifically bind antibodies of the particular animal species from whichthe anti-BoNT antibody is derived (e.g., an anti-species antibody).Thus, for example, where the capture agent is a human derived BoNT/Eantibody, the label agent may be a mouse anti-human IgG, i.e., anantibody specific to the constant region of the human antibody.

Other proteins capable of specifically binding immunoglobulin constantregions, such as streptococcal protein A or protein G are also used asthe labeling agent. These proteins are normal constituents of the cellwalls of streptococcal bacteria. They exhibit a strong non immunogenicreactivity with immunoglobulin constant regions from a variety ofspecies (see generally Kronval, et al., (1973)J. Immunol.,111:1401-1406, and Akerstrom, et al., (1985) J. Immunol., 135:2589-2542,and the like).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,analyte, volume of solution, concentrations, and the like. Usually, theassays are carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 5° C. to 45° C.

1) Non Competitive Assay Formats.

Immunoassays for detecting BoNT neurotoxins (e.g. BoNT serotypes and/orsubtypes) are, in certain embodiments, either competitive ornoncompetitive. Noncompetitive immunoassays are assays in which theamount of captured analyte (in this case, BoNT polypeptide) is directlymeasured. In one preferred “sandwich” assay, for example, the captureagent (e.g., an anti-BoNT antibody) is bound directly or indirectly to asolid substrate where it is immobilized. These immobilized anti-BoNTantibodies capture BoNT polypeptide(s) present in a test sample (e.g., ablood sample). The BoNT polypeptide(s) thus immobilized are then boundby a labeling agent, e.g.; an anti-BoNT/E antibody bearing a label.Alternatively, the second antibody may lack a label, but it may, inturn, be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. Free labeled antibodyis washed away and the remaining bound labeled antibody is detected(e.g., using a gamma detector where the label is radioactive).

2) Competitive Assay Formats.

In competitive assays, the amount of analyte (e.g., BoNT/E) present inthe sample is measured indirectly by measuring the amount of an added(exogenous) analyte displaced (or competed away) from a capture agent(e.g., anti-BoNT/E antibody) by the analyte present in the sample. Forexample, in one competitive assay, a known amount of BoNT/E is added toa test sample with an unquantified amount of BoNT/E, and the sample iscontacted with a capture agent, e.g., an anti-BoNT/E antibody thatspecifically binds BoNT/E. The amount of added BoNT/E that binds to theanti-BoNT/E-neutralizing antibody is inversely proportional to theconcentration of BoNT/E present in the test sample.

The anti-BoNT/E antibody can be immobilized on a solid substrate. Theamount of BoNT/E bound to the anti-BoNT/E antibody is determined eitherby measuring the amount of BoNT/E present in a BoNT/E-anti-BoNT/Eantibody complex, or alternatively by measuring the amount of remaininguncomplexed BoNT/E.

B) Reduction of Non Specific Binding.

One of skill will appreciate that it is often desirable to reduce nonspecific binding in immunoassays and during analyte purification. Wherethe assay involves, for example BoNT/E polypeptide(s),BoNT/E-neutralizing antibody, or other capture agent(s) immobilized on asolid substrate, it is desirable to minimize the amount of non specificbinding to the substrate. Means of reducing such non specific bindingare well known to those of skill in the art. Typically, this involvescoating the substrate with a proteinaceous composition. In particular,protein compositions such as bovine serum albumin (BSA), nonfat powderedmilk, and gelatin are widely used.

C) Substrates.

As mentioned above, depending upon the assay, various components,including the BoNT polypeptide(s), anti-BoNT antibodies, etc., areoptionally bound to a solid surface. Many methods for immobilizingbiomolecules to a variety of solid surfaces are known in the art. Forinstance, the solid surface may be a membrane (e.g., nitrocellulose), amicrotiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube(glass or plastic), a dipstick (e.g., glass, PVC, polypropylene,polystyrene, latex, and the like), a microcentrifuge tube, or a glass,silica, plastic, metallic or polymer bead. The desired component may becovalently bound, or noncovalently attached through nonspecific bonding.

A wide variety of organic and inorganic polymers, both natural andsynthetic may be employed as the material for the solid surface.Illustrative polymers include polyethylene, polypropylene,poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethyleneterephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidenedifluoride (PVDF), silicones, polyformaldehyde, cellulose, celluloseacetate, nitrocellulose, and the like. Other materials which may beemployed, include paper, glasses, ceramics, metals, metalloids,semiconductive materials, cements or the like. In addition, substancesthat form gels, such as proteins (e.g., gelatins), lipopolysaccharides,silicates, agarose and polyacrylamides can be used. Polymers which formseveral aqueous phases, such as dextrans, polyalkylene glycols orsurfactants, such as phospholipids, long chain (12-24 carbon atoms)alkyl ammonium salts and the like are also suitable. Where the solidsurface is porous, various pore sizes may be employed depending upon thenature of the system.

In preparing the surface, a plurality of different materials may beemployed, e.g., as laminates, to obtain various properties. For example,protein coatings, such as gelatin can be used to avoid non specificbinding, simplify covalent conjugation, enhance signal detection or thelike.

If covalent bonding between a compound and the surface is desired, thesurface will usually be polyfunctional or be capable of beingpolyfunctionalized. Functional groups which may be present on thesurface and used for linking can include carboxylic acids, aldehydes,amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto various surfaces is well known and is amply illustrated in theliterature. See, for example, Immobilized Enzymes, Ichiro Chibata,Halsted Press, New York, 1978, and Cuatrecasas, (1970) J. Biol. Chem.245 3059.

In addition to covalent bonding, various methods for noncovalentlybinding an assay component can be used. Noncovalent binding is typicallynonspecific absorption of a compound to the surface. Typically, thesurface is blocked with a second compound to prevent nonspecific bindingof labeled assay components. Alternatively, the surface is designed suchthat it nonspecifically binds one component but does not significantlybind another. For example, a surface bearing a lectin such asconcanavalin A will bind a carbohydrate containing compound but not alabeled protein that lacks glycosylation. Various solid surfaces for usein noncovalent attachment of assay components are reviewed in U.S. Pat.Nos. 4,447,576 and 4,254,082.

D) Other Assay Formats

BoNT polypeptides or anti-BoNT antibodies (e.g. BoNT/E neutralizingantibodies) can also be detected and quantified by any of a number ofother means well known to those of skill in the art. These includeanalytic biochemical methods such as spectrophotometry, radiography,electrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, and the like, and various immunological methods such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, and the like.

Western blot analysis and related methods can also be used to detect andquantify the presence of BoNT polypeptides in a sample. The techniquegenerally comprises separating sample products by gel electrophoresis onthe basis of molecular weight, transferring the separated products to asuitable solid support, (such as a nitrocellulose filter, a nylonfilter, or derivatized nylon filter), and incubating the sample with theantibodies that specifically bind either the BoNT polypeptide. Theantibodies specifically bind to the biological agent of interest on thesolid support. These antibodies are directly labeled or alternativelyare subsequently detected using labeled antibodies (e.g., labeled sheepanti-human antibodies where the antibody to a marker gene is a humanantibody) which specifically bind to the antibody which binds the BoNTpolypeptide.

Other assay formats include liposome immunoassays (LIAs), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.,(1986) Amer. Clin. Prod. Rev. 5:34-41).

E) Labeling of Anti-BoNT Anti-BoNT/E) Antibodies.

Anti-BoNT antibodies can be labeled by any of a number of methods knownto those of skill in the art. Thus, for example, the labeling agent canbe, e.g., a monoclonal antibody, a polyclonal antibody, a protein orcomplex such as those described herein, or a polymer such as an affinitymatrix, carbohydrate or lipid. Detection proceeds by any known method,including immunoblotting, western analysis, gel-mobility shift assays,tracking of radioactive or bioluminescent markers, nuclear magneticresonance, electron paramagnetic resonance, stopped-flow spectroscopy,column chromatography, capillary electrophoresis, or other methods whichtrack a molecule based upon an alteration in size and/or charge. Theparticular label or detectable group used in the assay is not a criticalaspect of the invention. The detectable group can be any material havinga detectable physical or chemical property. Such detectable labels havebeen well-developed in the field of immunoassays and, in general, anylabel useful in such methods can be applied to the present invention.Thus, a label is any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include magneticbeads (e.g. Dynabeads™), fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., LacZ, CAT, horse radishperoxidase, alkaline phosphatase and others, commonly used as detectableenzymes, either as marker gene products or in an ELISA), andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on the sensitivity required, ease of conjugation of thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to an anti-ligand (e.g., streptavidin) moleculewhich is either inherently detectable or covalently bound to a signalsystem, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence, e.g., by microscopy,visual inspection, via photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers andthe like. Similarly, enzymatic labels may be detected by providingappropriate substrates for the enzyme and detecting the resultingreaction product. Finally, simple colorimetric labels may be detectedsimply by observing the color associated with the label. Thus, invarious dipstick assays, conjugated gold often appears pink, whilevarious conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence ofBoNT peptides. In this case, antigen-coated particles are agglutinatedby samples comprising the target antibodies. In this format, none of thecomponents need be labeled and the presence of the target antibody isdetected by simple visual inspection.

IX. Pharmaceutical Compositions.

The BoNT-neutralizing antibodies of this invention are useful inmitigating the progression of botulisum produced, e.g., by endogenousdisease processes or by chemical/biological warfare agents. Typicallycompositions comprising one or preferably two or more differentantibodies are administered to a mammal (e.g., to a human) in needthereof.

We have discovered that particularly efficient neutralization of abotulism neurotoxin (BoNT) subtype is achieved by the use ofneutralizing antibodies that bind two or more subtypes of the particularBoNT serotype with high affinity. In certain embodiments, this can beaccomplished by using two or more different antibodies directed againsteach of the subtypes and/or neutralizing antibodies that bind two ormore BoNT subtypes (e.g., BoNT/A1, BoNT/A2, BoNT/A3, etc.) with highaffinity.

It was also a surprising discovery that when one starts combiningneutralizing antibodies that the potency of the antibody combinationincreases dramatically. This increase makes it possible to generate abotulinum antibody compositions of the required potency for therapeuticuse. It was also surprising that as one begins combining two and threemonoclonal antibodies, the particular BoNT epitope that is recognizedbecomes less important. Thus, in certain embodiments, this inventioncontemplates compositions comprising at least two, more preferably atleast three high affinity antibodies that bind non-overlapping epitopeson the BoNT.

In certain embodiments, this invention contemplates compositionscomprising two or more, preferably three or more different antibodiesselected from the group consisting of 2A10, 3E1, 3E2, 3E3, 3E4, 3E4.1,3E5, 3E6, 3E6.1, 4E11, 4E13, 4E16, 4E16.1, 4E17, 4E17.1, n A12, 6A12,B1.1, B6, B6.1, B8, B8.1, B11, B11C3, B11E8, B12, B12.1, B12.2, 1B18,2B18.1, 4B19, and 1B22, an/or antibodies comprising one or more CDRsfrom these antibodies, and/or one or more antibodies comprising mutantsof these antibodies.

The BoNT-neutralizing antibodies of this invention are useful forparenteral, topical, oral, or local administration, such as by aerosolor transdermally, for prophylactic and/or therapeutic treatment. Thepharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. For example,unit dosage forms suitable for oral administration include powder,tablets, pills, capsules and lozenges. The antibodies comprising thepharmaceutical compositions of this invention, when administered orally,are preferably protected from digestion. This is typically accomplishedeither by complexing the antibodies with a composition to render themresistant to acidic and enzymatic hydrolysis or by packaging theantibodies in an appropriately resistant carrier such as a liposome.Means of protecting proteins from digestion are well known in the art.

The pharmaceutical compositions of this invention are particularlyuseful for parenteral administration, such as intravenous administrationor administration into a body cavity or lumen of an organ. Thecompositions for administration will commonly comprise a solution of oneor more BoNT-neutralizing antibody dissolved in a pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of BoNT/A-neutralizing antibody in these formulations canvary widely, and will be selected primarily based on fluid volumes,viscosities, body weight and the like in accordance with the particularmode of administration selected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenousadministration would be about 0.1 to 10 mg per patient per day. Dosagesfrom about 1 mg up to about 200 mg per patient per day can be used.Methods for preparing parenterally administrable compositions will beknown or apparent to those skilled in the art and are described in moredetail in such publications as Remington's Pharmaceutical Science, 15thed., Mack Publishing Company, Easton, Pa. (1980).

The compositions containing the BoNT-neutralizing antibodies of thisinvention or a cocktail thereof are generally administered fortherapeutic treatments. Preferred pharmaceutical compositions areadministered in a dosage sufficient to neutralize (mitigate oreliminate) the BoNT toxin(s) (i.e., reduce or eliminate a symptom ofBoNT poisoning (botulism)). An amount adequate to accomplish this isdefined as a “therapeutically effective dose.” Amounts effective forthis use will depend upon the severity of the disease and the generalstate of the patient's health.

Single or multiple administrations of the compositions may beadministered depending on the dosage and frequency as required andtolerated by the patient. In any event, the composition should provide asufficient quantity of the antibodies of this invention to effectivelytreat the patient.

X. Kits For Diagnosis or Treatment.

In another embodiment, this invention provides for kits for thetreatment of botulism or for the detection/confirmation of a Clostridiumbotulinum infection. Kits will typically comprise one or more anti-BoNTantibodies (e.g., BoNT-neutralizing antibodies for pharmaceutical use)of this invention. For diagnostic purposes, the antibody(s) canoptionally be labeled. In addition the kits will typically includeinstructional materials disclosing means of use BoNT-neutralizingantibodies in the treatment of symptoms of botulism. The kits may alsoinclude additional components to facilitate the particular applicationfor which the kit is designed. Thus, for example, where a kit containsone or more anti-BoNT antibodies for detection of diagnosis of BoNTsubtype, the antibody can be labeled, and the kit can additionallycontain means of detecting the label (e.g. enzyme substrates forenzymatic labels, filter sets to detect fluorescent labels, appropriatesecondary labels such as a sheep anti-human antibodies, or the like).The kits may additionally include buffers and other reagents routinelyused for the practice of a particular method. Such kits and appropriatecontents are well known to those of skill in the art.

In certain embodiments, kits provided for the treatment of botulisumcomprise one or more BoNT neutralizing antibodies. The antibodies can beprovided separately or mixed together. Typically the antibodies will beprovided in a sterile pharmacologically acceptable excipient. In certainembodiments, the antibodies can be provided pre-loaded into a deliverydevice (e.g., a disposable syringe).

The kits can optionally include instructional materials teaching the useof the antibodies, recommended dosages, contraindications, and the like.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1.-31. (canceled)
 32. An isolated antibody, or antigen-binding fragmentthereof, that specifically binds an epitope of a Botulinum neurotoxin(BoNT), wherein the antibody or antigen-binding fragment comprises: a) aV_(H) CDR1 comprising the amino acid sequence of SEQ ID NO:166; b) aV_(H) CDR2 comprising the amino acid sequence of SEQ ID NO:168; c) aV_(H) CDR3 comprising the amino acid sequence of SEQ ID NO:170; d) aV_(L) CDR1 comprising the amino acid sequence of SEQ ID NO:271; e) aV_(L) CDR2 comprising the amino acid sequence of SEQ ID NO:273; and f) aV_(L) CDR3 comprising the amino acid sequence of SEQ ID NO:275.
 33. Acomposition comprising the isolated antibody or antigen-binding fragmentof claim 32 and a pharmaceutically acceptable carrier.
 34. The isolatedantibody or antigen-binding fragment of claim 32, wherein said antibodyis a humanized antibody.
 35. The isolated antibody or antigen-bindingfragment of claim 34, wherein said antibody is a human antibody.
 36. Theisolated antibody or antigen-binding fragment of claim 32, wherein saidantibody is a single chain Fv (scFv), Fab, (Fab′)₂ or (scFv′)₂.
 37. Theisolated antibody or antigen-binding fragment of claim 32, wherein saidantibody is an IgG.
 38. A method of specifically binding a botulinumneurotoxin in a mammal, said method comprising administering to saidmammal the isolated antibody or antigen-binding fragment of claim 32.39. A kit for specifically binding a Botulinum neurotoxin, said kitcomprising: a composition according to claim
 32. 40. The isolatedantibody or antigen-binding fragment of claim 32, wherein said antibodycomprises: a heavy chain comprising the amino acid sequence of SEQ IDNO:52.
 41. A composition comprising the isolated antibody orantigen-binding fragment of claim 40 and a pharmaceutically acceptablecarrier.
 42. The isolated antibody or antigen-binding fragment of claim40, wherein said antibody is a humanized antibody.
 43. The isolatedantibody or antigen-binding fragment of claim 41, wherein said antibodyis a human antibody.
 44. The isolated antibody or antigen-bindingfragment of claim 40, wherein said antibody is a single chain Fv (scFv),Fab, (Fab)₂ or (scFv′)₂.
 45. The isolated antibody or antigen-bindingfragment of claim 40, wherein said antibody is an IgG.
 46. A method ofspecifically binding a botulinum neurotoxin in a mammal, said methodcomprising administering to said mammal the isolated antibody orantigen-binding fragment of claim
 40. 47. A kit for specifically bindinga Botulinum neurotoxin, said kit comprising: a composition according toclaim
 40. 48. The isolated antibody or antigen-binding fragment of claim32, wherein said antibody comprises: a light chain comprising the aminoacid sequence of SEQ ID NO:67.
 49. The isolated antibody orantigen-binding fragment of claim 32, wherein said antibody comprises:a) a heavy chain comprising the amino acid sequence of SEQ ID NO:52; andb) a light chain comprising the amino acid sequence of SEQ ID NO:67. 50.A composition comprising the isolated antibody or antigen-bindingfragment of claim 49 and a pharmaceutically acceptable carrier.
 51. Theisolated antibody or antigen-binding fragment of claim 49, wherein saidantibody is a humanized antibody.
 52. The isolated antibody orantigen-binding fragment of claim 51, wherein said antibody is a humanantibody.
 53. The isolated antibody or antigen-binding fragment of claim49, wherein said antibody is a single chain Fv (scFv), Fab, (Fab)₂ or(scFv′)₂.
 54. The isolated antibody or antigen-binding fragment of claim49, wherein said antibody is an IgG.
 55. A method of specificallybinding a botulinum neurotoxin in a mammal, said method comprisingadministering to said mammal the isolated antibody or antigen-bindingfragment of claim
 49. 56. A kit for specifically binding a Botulinumneurotoxin, said kit comprising: a composition according to claim 49.